Ultrafast(Nonlinear(Spectroscopy(of(Red(Fluorescent(Proteins(!
by!
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Patrick!Eugene!Konold!
B.S.!University!of!Minnesota,!2008!
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A!thesis!submitted!to!the!
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Faculty!of!the!Graduate!School!of!the!
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University!of!Colorado!in!partial!fulfillment!
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of!the!requirement!for!the!degree!of!
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Doctor!of!Philosophy!
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Department!of!Chemistry!and!Biochemistry!
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2015!
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This!thesis!entitled:!
Ultrafast!Nonlinear!Spectroscopy!of!Red!Fluorescent!Proteins!
written!by!Patrick!Eugene!Konold!
has!been!approved!for!the!Department!of!Chemistry!and!Biochemistry!
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Ralph!Jimenez!
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Steven!Cundiff!
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Date__________________!
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The!final!copy!of!this!thesis!has!been!examined!by!the!signatories,!and!we!find!that!
both!the!content!and!the!form!meet!acceptable!presentation!standards!of!scholarly!
work!in!the!above!mentioned!discipline

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Konold,!Patrick!Eugene!(Ph.D.,!Physical!Chemistry,!Department!of!Chemistry!and!
Biochemistry)!
Ultrafast(Nonlinear(Spectroscopy(of(Red(Fluorescent(Proteins(
Thesis!directed!by!Professor!Ralph!Jimenez!
RedVemitting!homologues!(RFPs)!of!the!native!Green!Fluorescent!Protein!
(GFP)!with!emission!wavelengths!beyond!650!nm!are!desirable!probes!for!in#vivo#
imaging!experiments.!!They!offer!the!potential!for!deeper!tissue!penetration!and!
lower!background!scatter!given!a!cleaner!spectral!window.!!However,!bioimaging!
applications!are!hindered!by!poor!photophysics!(e.g.!low!fluorescence!quantum!
yield,!high!photobleaching),!which!limits!experimental!resolution!and!represents!a!
significant!obstacle!towards!utilization!for!low!copyVnumber,!longVduration!imaging!
applications.!!In!this!thesis,!a!variety!of!femtosecond!nonlinear!electronic!
spectroscopies!were!employed!jointly!with!siteVdirected!mutagenesis!to!investigate!
the!photophysical!properties!of!RFPs.!!In!one!study,!the!molecular!mechanism!of!red!
emission!was!pursued!in!two!notable!RFPs,!mPlum!and!TagRFP675.!!Solvation!
dynamics!observed!with!timeVresolved!transient!grating!spectroscopy!were!
interpreted!with!the!aid!of!molecular!dynamics!simulations!to!indicate!that!their!
redVemission!is!correlated!with!the!ability!of!specific!chromophoreVsidechain!
hydrogenVbonding!interactions!to!interconvert!between!direct!and!waterVmediated!
states.!!In!a!second!set!of!studies,!twoVdimensional!double!quantum!coherence!
spectroscopy!was!used!to!probe!the!electronic!transitions!of!mPlum.!!It!was!
discovered!that!it!displayed!a!response!distinctly!different!from!an!organic!dye!in!
bulk!solvent.!!Modeling!indicate!of!these!spectra!indicate!the!spectral!features!may!
be!attributed!to!the!existence!of!multiple!highVlying!(n>1)!excited!states.!!The!results!

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provide!new!insight!into!the!electronic!structure!of!these!widely!used!fluorescent!
probes.!!!
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Dedication(
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I!dedicate!this!thesis!to!my!family!and!friends!who!inspired!me!to!think!big!and!
realize!my!full!potential!no!matter!the!odds!!!
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Acknowledgements((
Several!people!were!instrumental!to!both!my!personal!and!professional!
enrichment!throughout!this!journey.!!I!am!very!grateful!to!all!I!have!encountered!
during!my!time!at!CU!and!in!the!greater!Boulder!area.!!!!!
First,!I!must!thank!my!advisor,!Professor!Ralph!Jimenez,!for!helping!me!
embrace!the!discipline!of!biophysics!and!convincing!me!that!proteins!are!not!all!that!
scary.!!Orchestrating!interdisciplinary!research!is!no!easy!task!and!our!success!is!a!
testament!to!his!qualification!as!both!a!scientist!and!a!mentor.!!Moreover,!his!high!
expectations!have!helped!expedite!my!scientific!development!and!acquire!the!
problemVsolving!skills!and!confidence!necessary!to!foster!success!in!my!looming!
professional!career.!!!!!
It’s!very!safe!to!say!I!would!not!have!pursued!a!graduate!degree!without!
inspiration!from!Professor!Aaron!Massari!at!the!University!of!Minnesota.!!I!
appreciate!his!willingness!to!lend!laboratory!carte#blanche!to!an!inexperienced,!yet!
relentlessly!curious!and!ambitious,!chemistry!student.!!This!opportunity!laid!the!
foundation!for!my!current!academic!success!and!I!am!eternally!thankful.!!!
I!must!also!recognize!present!and!past!members!of!the!Jimenez!group!who!
were!a!great!help!over!these!years.!!!Thanks!to!Jennifer!Lubbeck!for!recruiting!me!to!
the!group.!!Hairong!Ma!for!keeping!me!busy!through!the!first!broken!laser.!!Linda!
Schwall!and!Pia!Friis!for!doing!a!whole!lot!of!pipetting/incubating/centrifuging!in!
the!preparation!of!countless!protein!samples!allowing!me!to!focus!on!development!

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of!spectroscopic!experimentation,!and!also!for!introducing!me!to!the!basics!of!
molecular!biology.!!Kevin!Dean!for!useful!scientific!discussions!and!being!an!outlet!
when!things!weren’t!going!so!smooth.!!!
JILA!is!a!wonderful!environment!to!do!scientific!research!and,!in!general,!a!
great!place!to!work.!!I!especially!recognize!the!support!staff!who!I’ve!solicited!
uninvited!countless!times!over!these!past!years.!!A!special!thanks!is!necessary!to!
Hans!Green,!Todd!Asnicar,!and!Kim!Hagen!(instrument!shop),!JR!Raith!and!Jim!
McKown!(computing),!Terry!Brown!(electronics),!and!Brian!Lynch!(purchasing).!!
Their!eagerness!to!lend!assistance!(at!almost!any!hour)!demonstrates!a!true!passion!
for!their!work!and!is!a!critical!advantage!of!conducting!research!here!at!JILA.!!!
Several!scientific!collaborators!were!instrumental!in!the!realization!of!this!
project.!!Thanks!to!Cundiff!group!members!Alan!Bristow!and!Galan!Moody!for!
helping!troubleshoot!the!sometimesVornery!JILA!MONSTR.!!Prem!Chapagain!and!
Bernard!Gerstman!for!carrying!out!MD!simulations!that!greatly!helped!with!the!
interpretation!of!our!experimental!results.!!KM!group!members!Dan!Adams!and!
Matt!Seaberg!for!assistance!with!our!brief!foray!into!adaptive!pulse!shaping.!!!
Finally,!I!must!acknowledge!nonscientific!friends!and!acquaintances!that!
were!instrumental!in!keeping!my!mind!fresh!throughout!this!effort!and!are!perhaps!
most!responsible!my!academic!success!to!date.!!For!the!record!guys,!I!do!not!operate!
a!laser!light!show.!!I!owe!a!large!debt!of!gratitude!to!my!friends!Mike!(and!Jenna)!
Belaen!and!David!Wambeke!dating!back!to!earlier!times!in!Minneota.!!Our!
achievements!are!empirical!proof!that,!with!a!proper!mindset,!anything!is!possible.!!

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Sam,!Tiff,!and!Alena!Kasanicky!for!offering!the!epitome!of!friendship.!!Thank!you!to!
my!golf!companions.!!You!know!who!you!are,!and!you!probably!owe!me!money.!!
Thanks!to!the!entire!Murphy’s/Snugbutter!soccer!team!for!introducing!me!to!a!new!
game!and!giving!me!a!chance!to!run!around!in!the!fresh!air!in!between!moments!of!
scientific!rigor.!!
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ix!
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Table(of(Contents(!
Chapter!
I.

Introduction............................................................................................................................1!
Spectral!properties!of!Fluorescent!Proteins...........................................................................................1!
Nonlinear!Femtosecond!Spectroscopy.....................................................................................................!7!
Survey!of!Nonlinear!Optical!Methods.....................................................................................................18!!
Thesis!Objectives.............................................................................................................................................!26!

II.

Experimental!Methods....................................................................................................!28!
Description!of!Light!Source........................................................................................................................!28!
Ultrafast!Pulse!Compression......................................................................................................................!36!
JILA!MONSTR!Nonlinear!Optical!Platform...........................................................................................!52!
Data!Acquisition!Procedures......................................................................................................................60!
Sample!Preparation!Protocol.....................................................................................................................!64!
Auxiliary!Experimental!Details..................................................................................................................67!

III.

Hydrogen!Bond!Flexibility!Correlates!with!!
Stokes!Shift!in!mPlum!Variants...................................................................................71!
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Introduction.......................................................................................................................................................71!
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Experimental!Methods..................................................................................................................................!74!
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Results..................................................................................................................................................................!77!
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Discussion...........................................................................................................................................................97!
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Conclusions.......................................................................................................................................................101!

IV.

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The!Origin!of!Extended!Stokes!Shift!of!TagRFP675.........................................103!
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Introduction.....................................................................................................................................................103!
Experimental!Methods................................................................................................................................106!
Results................................................................................................................................................................109!
Discussion.........................................................................................................................................................118!

Conclusions.......................................................................................................................................................122!

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V.

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VI.!
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Excited!State!Electronic!Structure!of!mPlum!Revealed!!
by!TwoVDimensional!Double!Quantum!Coherence!Spectroscopy.............124!
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Introduction.....................................................................................................................................................124!
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Experimental!Methods................................................................................................................................127!
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Results................................................................................................................................................................129!
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Discussion.........................................................................................................................................................142!
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Conclusions......................................................................................................................................................148!

Conclusions!and!Future!Outlook..............................................................................!150!
Future!Outlook...............................................................................................................................................!150!!

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Bibliography............................................................................................................................................153!
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Appendix...................................................................................................................................................165!!
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A. Data!Acquisition!Software..................................................................................................................................!165!
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B. Data!Processing!Software...................................................................................................................................!166!

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C.
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Data!Modeling!Software......................................................................................................................................172!

D. JILA!MONSTR!Calibration!Procedures..........................................................................................................172!

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E.

SpectrallyVResolved!Signal!Detection............................................................................................................176!

F.

Additional!Experimental!Considerations.....................................................................................................177!
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List(of(Tables(!
Table!
3.1.
3.2.
3.3.
3.4.
3.5.
3.6.

Excitation!center!position!and!FWHM!bandwidth!used!in!SRTG!
measurements!
!
Steady!state!absorption!and!emission!data!for!each!mutant!
Summary!of!SRTG!fitting!results!following!fitting!the!raw!data!to!a!sum!of!
two!Gaussians!
!
Fitting!error!analysis!for!multiexponential!fits!to!SE!band!shifts!
!
SRTG!amplitude!decays!of!GSB!and!SE!bands!

4.1.

Survival!times!calculated!for!the!direct!hydrogen!bond!molecular!
configuration!and!for!the!waterVmediated!hydrogen!bond!configurations!
!
Probability!of!I65Vwater!hydrogen!bond!without!simultaneous!16Vwater!
hydrogen!bond!from!MD!simulation!for!each!mutant!
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HydrogenVbonding!to!chromophore!from!neighboring!sidechains!in!terms!of!
time!fraction!as!calculated!from!MD!simulations!
!
Steady!state!absorption!and!emission!data!for!all!TagRFP675!mutants!

4.2.

Fitting!parameters!assuming!exponential!shift!of!SE!band!

4.3.

Fitting!error!analysis!for!multiexponential!fits!to!SE!band!shifts!

4.4.

Amplitude!decay!fits!of!SE!band!

4.5.

HydrogenVbonding!to!the!chromophore!from!neighboring!sidechains!
presented!in!terms!of!time!fraction!as!calculated!from!MD!simulations!!!

5.1.!

Energy!values!(cmV1)!for!various!electronic!level!structures!used!to!model!
2D2Q!experimental!data!

3.7.
3.8.

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List(of(Figures(
Figure!
1.1.!

Representative!monomeric!fluorescent!protein!structure!!

1.2.!

Chemical!structure!of!a!chromophore!characteristic!of!a!green!and!redV
emitting!fluorescent!protein!

1.3.!

Guidelines!for!interpreting!doubleVsided!Feynman!diagrams!used!to!
described!nonlinear!lightVmatter!interactions!

1.4.!

Third!order!nonlinear!optical!response!functions!for!a!threeVlevel!system!

1.5.!

The!time!evolution!of!an!electronic!transition!frequency!in!the!condensed!
phase!

1.6.!

An!electronic!absorption!lineshape!broken!into!its!inhomogeneous!and!
homogeneous!components!

1.7.!

Beam!geometry!and!pulse!sequence!for!a!transient!absorption!experiment!

1.8.!

Beam!geometry!and!pulse!sequence!for!a!typical!transient!grating!
experiment!

1.9.!

Third!order!responses!for!a!transient!grating!experiment!of!a!threeVlevel!
system!

1.10! Evolution!of!the!collective!phase!of!the!excited!volume!throughout!a!photon!
echo!spectroscopy!experiment!
1.11.! Beam!geometry!and!pulse!sequence!for!a!stimulated!photon!echo!
spectroscopy!experiment!
1.12.! Expected!twoVdimensional!spectral!evolution!of!a!condensed!twoVlevel!
electronic!system!
1.13.! Expected!twoVdimensional!spectral!evolution!for!the!case!of!electronic!
population!transfer!
2.1.!!! Outline!of!the!light!source!used!for!timeVresolved!experimentation!
2.2.!

Grism!pair!stretcher!used!to!chirp!oscillator!output!seeding!WyvernV20!
regenerative!amplifier!

2.3.!

Beam!diagram!of!the!KM!Labs!WyvernV20!regenerative!amplifier!light!source!

2.4.!

NOPA!beam!diagram!used!to!generate!broadband!femtosecond!pulses!in!the!
visible!spectral!region!

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2.5.!

Typical!NOPA!spectral!output!with!various!white!light!chirp!as!a!function!of!
pumpVseed!delay!

2.6.!

Prism!pair!compressor!beam!path!used!to!compress!NOPA!output!

2.7.!

Dispersion!curves!for!Venteon!DCM10!chirped!mirror!pair!

2.8!

Beam!path!diagram!for!doubleVpass!alignment!of!the!chirped!mirror!pair!

2.9.!

TG!FROG!measured!from!the!nonresonant!signal!of!neat!benzene!collected!at!
sample!position!

2.10.! Beam!diagram!of!adaptive!pulse!shaper!
2.11.! Freezing!algorithm!utilized!for!pulse!compression!
2.12.! Applied!phase!obtained!upon!optimization!with!freezing!algorithm!
2.13.! TG!FROG!before!and!after!optimization!with!freezing!algorithm!
2.14.! Integrated!TG!FROG!before!and!after!optimization!
2.15.! Nile!Blue!in!acetonitrile!TG!spectrum!following!compression!with!freezing!
algorithm!
2.16.! Nile!blue!TG!data!at!early!delay!and!respective!frequency!slices!with!SLM!
power!on!and!off!
2.17.! Frequency!and!temporal!representations!in!the!pulse!overlap!region!of!the!
nile!blue!TG!spectrum!with!SLM!on!and!off!
2.18.! Spectral!envelope!following!application!of!a!sinusoidal!phase!on!resonant!
and!nonresonant!samples!
2.19.! Photographs!of!light!reflected!off!the!SLM!with!power!on!and!off!
2.20.! Spectral!envelope!and!corresponding!temporal!spectrum!for!a!second!SLM!
2.21.! Spectral!envelope!of!spatiallyVfiltered!nile!blue!FWM!signal!
2.22.! CAD!drawings!of!JILA!MONSTR!assembly!and!beam!paths!
2.23.! Photographs!of!JILA!MONSTR!
2.24.! Beam!path!outside!of!JILA!MONSTR!
2.25.! Field!correlations!for!various!beam!combinations!collected!at!the!replica!
focus!position!
2.26.! Stepping!sequence!for!XVaxis!phase!cycling!in!nonrephasing!and!rephasing!
data!collection!scans!

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2.27.! Photographs!of!low!and!high!yield!fluorescent!protein!cell!pellet!
2.28.! Overview!of!Quikchange!siteVdirected!mutagenesis!protocol!
2.29.! Cell!used!to!contain!liquid!samples!for!spectroscopic!experimentation!
2.30.! Photographs!of!spinning!sample!cell!
2.31.! Phase!stability!with!and!without!spinning!sample!cell!!
3.1.!

Chemical!structure!of!mature!mPlum!chromophore!

3.2.!

SpectrallyVresolved!transient!grating!spectra!and!stimulated!emission!peak!
positions!for!mPlum!and!the!I65L!mutant!

3.3.!

SpectrallyVresolved!transient!absorption!spectrum!of!mPlum!

3.4.!

Stimulated!emission!(SE)!peak!positions!for!remaining!mPlum!mutants!

3.5.!

Simulated!SRTG!data!with!and!without!application!of!a!finite!excitation!
bandwidth!and!excitation!absorption!response!

3.6.!

Unique!hydrogen!bond!conformations!identified!in!MD!simulations!of!mPlum!

3.7.!

Water!occupancy!within!3!Å!of!chromophore!over!a!40!ns!time!trajectory!for!
mPlum!and!variants!

3.8.!

MD!time!series!trajectories!for!all!mPlum!mutants!

3.9.!

MD!time!series!trajectory!for!the!mPlum!E16L!mutant!

3.10.! Histograms!of!the!probability!for!a!16V65!separation,!Δr,!to!occur!during!the!
MD!simulations!
3.11.! Survival!functions!calculated!from!the!MD!simulations!for!the!direct!and!
waterVmediated!hydrogen!bonds!
3.12.! Calculated!chromophore!RMSF!for!each!mutant!
4.1.!

Specific!hydrogen!bond!interactions!identified!in!crystal!structures!of!
TagRFP675!attributed!to!its!farVred!emission!

4.2.!

SRTG!spectra!of!TagRFP675,!TagRFP675!Q41M,!mKate,!and!mKate!M41Q!

4.3.!

SE!band!peak!positions!extracted!from!double!Gaussian!fitting!of!SRTG!
spectra!and!multiexponential!decay!fits!for!TagRFP675!and!mKate!M41Q!

4.4.!

SRTG!spectra!and!fitted!SE!band!positions!for!various!time!delays!of!
TagRFP675!F62A,!TagRFP675!Q106M,!TagRFP675!Q106M!F62A!

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4.5.!

Time!trajectories!of!the!Q41Vacylimine!carbonyl!distance!(Δr)!for!TagRFP675!
and!mKate!M41Q.!!Δr!refers!to!the!distance!between!the!sidechain!of!Q41!and!
the!NVacylimine!oxygen!of!F62!

4.6.!

Time!trajectories!of!the!S28VQ41!distance!(Δr)!for!TagRFP675!and!mKate!
M41Q.!!Δr!is!defined!as!the!distance!between!the!sidechains!of!S28!and!Q41!

4.7.!

Histograms!of!the!probability!for!the!Q41VF62!and!Q41VS28!separations,!Δr,!
to!occur!during!the!MD!simulations!
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5.1!

Excitation!pulse!sequence!used!to!generate!the!third!order!nonlinear!optical!
signal!

5.2.!

Steady!state!absorption!and!emission!spectra!of!mPlum!

5.3.!

Excitation!dependent!SRTA!spectra!of!mPlum!

5.4.!

Absolute!value!and!real!2D2Q!spectra!of!cresyl!violet!in!methanol!

5.5.!

Absolute!value!2D2Q!spectra!of!mPlum!and!cresyl!violet!

5.6.!

Excitation!dependent!absolute!value!2D2Q!spectra!of!mPlum!!

5.7.!

Electronic!level!structures!considered!for!modeling!of!2D2Q!data!

5.8.!

LightVmatter!interactions!representing!the!double!quantum!responses!of!a!1V
1V2!electronic!level!structure!

5.9.!

Simulated!absolute!value!2D2Q!spectra!for!two!independent!3LS!with!
opposite!anharmonicity!

5.10.! Simulated!absolute!2D2Q!spectra!of!two!independent!3LS!with!negative!and!
positive!anharmonicity!
5.11.! Simulated!2D2Q!spectra!of!a!1V1V2!level!structure!with![+!V]!anharmonicity!
5.12.! Simulated!2D2Q!spectra!of!a!1V1V2!level!structure!with!negative!and!positive!
anharmonicity!
5.13.! Simulated!absolute!value!2D2Q!response!of!the!1V2V1!level!structure!with!
negative!and!positive!anharmonicity!
5.14.! Absolute!value!2D2Q!spectra!of!mKate,!mKate!S158A,!and!mKate!S158C!!!
5.15.! mPlum!absolute!value!2D1Q!spectra!at!t2=0,!10,!and!75!ps!
5.16.! Cresyl!violet!absolute!value!2D1Q!spectra!at!t2=0,!150,!and!400!fs!
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!1!
Chapter(I(!
Introduction(!
1.1(Spectral(properties(of(Fluorescent(Proteins!(
Fluorescent!proteins!(FPs),!originally!derived!from!marine!organisms,!have!
revolutionized!cellular!imaging!experiments!and!become!an!invaluable!tool!for!
molecular!biology!research1.!!Manipulation!of!the!progenitor!Green!Fluorescent!
Protein!(GFP)!from!the!jellyfish!Aequorea#victoria!have!led!to!creation!of!monomeric!
variants!with!tunable!excitation!and!emission!and!serve!as!noninvasive,!genetically!
targetable!fluorescent!probes!of!subcellular!function2.!!To!better!facilitate!these!
advanced!applications,!nextVgeneration!FPs!with!enhanced!photophysical!properties!
are!highly!desirable!and!offer!the!opportunity!to!bolster!a!new!class!of!nanoscopic!
imaging!technologies.!!These!improvements!include!farVred!emitting!variants!with!
emission!wavelengths!beyond!650!nm,!improved!chromophore!maturation,!
increased!brightness,!reduced!photobleaching,!and!efficient!photomodulatability!to!
serve!advanced!applications!of!superresolution!nanoscopy3V6.!!Such!technology!has!
effectively!broken!the!diffraction!limit!barrier!and!has!the!potential!to!capture!
biological!activity!in!real!time!with!near!molecularVlimited!precision7.!!Ultrafast!
spectroscopy!has!been!utilized!to!explore!the!mechanisms!of!excitedVstate!proton!
transfer!in!GFP8.!!However,!no!thorough!investigation!of!red!fluorescent!protein!
(RFP)!photophysics!has!been!reported!to!date.!!
Development!of!RFPs!has!proceeded!along!two!primary!lineages.!!DsRed!was!
isolated!from!the!mushroom!coral!Discosoma#and!led!to!the!creation!of!a!series!of!

!2!
species!with!variable!emission!wavelengths!spanning!the!visible!spectral!region!
known!as!the!mFruits9V11.!!Another!source!of!development!was!eqFP578,!originally!
extracted!from!the!sea!anemone!Entacmaea#quadricolor,!spawned!the!development!
of!the!monomeric!derivatives!TagRFP!and!mKate12V13.!!To!date,!the!major!strategy!
for!RFP!improvement!has!relied!on!directed!evolution!of!these!parent!species!
typically!guided!by!the!standard!methods!of!structural!biology!and!protein!
engineering14V15.!!The!recent!invention!of!nanoscopic!imaging!applications!has!
prompted!the!need!for!more!versatile!photoactivatible!RFPs!with!high!fluorescence!
quantum!yield!and!photostability.!!Simultaneous!optimization!of!these!
photophysical!parameters!would!represent!a!transformative!advancement!in!RFP!
development.!!!
The!structural!architecture!of!a!FP!is!an!11Vstranded!βVbarrel!as!depicted!in!a!
rendering!of!the!crystal!structure!in!Figure!1.1.!!The!fluorescent!chromophore!is!
embedded!deeply!within!this!framework!and!is!presumed!largely!isolated!from!the!
surrounding!solvent!bath.!!Although,!defects!within!the!barrel!arising!throughout!
the!development!of!monomer!analogues!of!the!native!oligomeric!species!are!
suspected!as!entrance!pathways!for!molecular!oxygen!that!is!thought!to!hinder!their!
photostability16.!!!

!3!

a

b

!
Figure(1.1.((Representative!monomeric!fluorescent!protein!structure!with!(a)!side!
and!(b)!top!perspectives.!!The!fluorescent!chromophore!(green)!is!embedded!within!
an!11Vstranded!βVbarrel.!
The!fluorescent!p-hydroxybenzylideneimidazolinone chromophore isformed!autocatalytically!in!the!presence!of!molecular!oxygen!with!residues!65V67!
(SVWVG)17.!!The!conjugated!system!of!GFP!spans!two!ring!moieties,!phenoxy!and!
imidazolinone!as!illustrated!in!Figure!1.2.!!Upon!electronic!excitation,!electron!
density!shifts!from!the!phenoxy!ring!towards!the!imidazolinone18.!!A!common!
strategy!in!the!development!of!redVemitting!species!involves!extending!the!
conjugated!network!by!chemically!integrating!neighboring!sidechains!through!postV
translational!modification.!!A!classic!example!of!this!occurs!in!the!RFP!DsRed,!where!
an!NVacylimine!forms!autocatalytically!adjacent!to!the!imidazolinone!ring!and!
accounts!for!its!redVshift!excitation!and!emission!relative!to!GFP19.!!The!“DsRedVlike”!
chromophore!and!subsequent!variants!have!provided!the!most!fruitful!template!for!
RFP!development!to!date.

!4!
!
!

a

b
-

O

N

O

O

-

N

N

O

N

N

O

!
Figure(1.2.((Chemical!structure!of!the!chromophore!from!the!(a)!GFP!and!(b)!DsRed!
fluorescent!proteins.!!The!conjugated!system!is!highlighted!in!each!case.!
The!rich!photophysics!of!GFP!has!attracted!much!attention!in!the!past!decade.!!
It!was!initially!discovered!that!the!4’V!hydroxybenzylideneV2,3VdimethylV
imidazolinone!(HDBI)!chromophore!shows!remarkably!different!spectral!properties!
in!solution!versus!embedded!within!the!native!protective!βVbarrel!structure.!!Most!
notably,!the!1000Vfold!higher!fluorescence!efficiency!(QY=0.8,!lifetime=3.3!ns)!in!
GFP!versus!in!solution!suggests!that!the!surrounding!protein!matrix!must!play!a!
pivotal!role!in!preventing!nonradiative!deactivation!of!the!excited!chromophore20V21.!!
GFP!contains!two!absorption!bands!at!398!and!478!nm!and!a!single!emission!peak!at!
510!nm17.!!The!two!absorption!peaks!are!attributed!to!the!neutral!and!anionic!
protonation!states!of!the!chromophore.!!TimeVresolved!fluorescence!experiments!by!
Chattoraj!and!coworkers!first!implicated!Excited!State!Proton!Transfer!(ESPT)!as!
the!underlying!cause!of!GFPs!unique!emission8.!!The!precise!structural!fluctuations!
mediating!these!transfer!events!has!been!the!subject!of!several!timeVresolved!

!5!
optical!and!vibrational!studies!and!has!been!thoroughly!reviewed22.!!These!
investigations!collectively!point!towards!ESPT!occurring!through!an!intricate!
hydrogen!bond!network!involving!S205,!E222,!and!embedded!water!molecules.!!!
Recently,!Mathies!and!coworkers!identified!a!key!wagging!motion!responsible!for!
modulation!of!ESPT!in!the!neutral!chromophore23.!!Similar!ESPT!pathways!have!
been!uncovered!in!other!FPs24V25.!!It!is!speculated!that!these!unique!spectral!
properties!are!owed!to!preorganized!chromophoreVbath!interactions!with!the!
surrounding!protein!environment.!!!
More!complex!phototransformations!occur!in!several!other!FP!systems!
including!on/off!photoswitching,!photoactivation,!and!photoconversion25V27.!!This!
occurs!in!GFP!following!prolonged!excitation!at!400!nm!where!gradual!
accumulation!of!a!photoproduct!with!80!nm!redshifted!absorption!is!thought!due!to!
irreversible!oxidation!of!CO2!from!adjacent!E22228.!!Similar!photoactivity!is!
observed!in!other!proteins.!!For!instance,!rhodopsin!and!Photoactive!Yellow!Protein!
(PYP)!are!known!to!undergo!photoinduced!isomerization!and!ultrafast!spectroscopy!
has!been!used!extensively!to!unravel!their!respective!photocycles29V32.!!Perhaps,!the!
most!widely!studied!photoswitchable!FP!is!Dronpa,!derived!from!the!coral!
Pectiniidae,!which!exhibits!distinct!cis!and!trans!conformations,!in!the!light!and!dark!
states!identified!through!structural!characterization33V36.!!!It!was!speculated!that!
Dronpa!underwent!ESPT!similar!to!GFP!upon!its!discovery36.!!Optical!pumpV
vibrational!probe!experiments!by!Warren!et#al.!indicated!a!9!ps!isomerization!time!
constant!and!upon!vibrational!analysis,!imply!proton!transfer!is!thermally!driven!in!
the!ground!state.!!Moreover,!they!identify!a!weakening!hydrogen!bond!between!R66!

!6!
and!the!chromophore!carbonyl!that!may!initiate!the!ultimate!structural!
rearrangements!leading!to!photoisomerization37.!!However,!a!broader!
characterization!of!these!dynamics!is!unclear.!!Another!study!involving!Dronpa!and!
single!point!mutants!displayed!unique!viscosity!dependent!photochromism!not!
previously!observed!in!FPs38.!!They!hypothesized!that!a!mechanical!coupling!from!
the!protein!exterior!regulates!flexibility!near!the!buried!chromophore.!!It!also!
highlights!the!role!of!environmental!dynamics!in!the!apparent!tradeoff!between!
fluorescence!quantum!yield!and!photoswitchability39.!!The!advent!of!
superresolution!nanoscopic!imaging!methods!such!as!photoactivated!localization!
microscopy!(PALM)!and!stochastic!reconstruction!microscopy!(STORM)!has!
motivated!the!need!for!more!robust!redVemitting!photoactivatible!FPs4,!7,!40V41.!!
Photoactivatible!analogues!of!mCherry!and!TagRFP675!have!been!devised!to!
advance!the!utility!of!these!powerful!techniques42V43.!!
Alternatively,!specific!interactions!between!the!chromophore!and!local!
sidechains!are!thought!to!influence!FP!spectral!properties.!!mPlum!was!developed!
through!iterative!hypersomatic!mutation!of!mRFP,!from!the!progenitor!DsRed,!to!
achieve!a!Stokes!shift!of!59!nm!and!represents!one!of!the!farthest!red!emitting!
monomeric!FPs!discovered!to!date44.!!A!fluorescence!upconversion!study!by!Abbyad!
et#al.!identified!a!significant!dynamic!Stokes!shift!in!mPlum!that!was!distinct!from!
mRaspberry!and!mRFP!which!demonstrated!negligible!solvation!over!a!nanosecond!
time!window45.!!They,!along!with!a!later!crystallographic!study,!attributed!the!
extended!Stokes!shift!of!mPlum!to!a!specific!direct!hydrogen!bond!interaction!
between!the!E16!sidechain!and!NVacylimine!oxygen!of!I6546.!!It!is!thought!that!these!

!7!
interactions!facilitate!stabilization!of!the!excited!state!by!a!combination!of!indirect!
modulation!of!hydrogen!bonds!at!other!positions!across!the!chromophore!as!well!as!
acting!to!restrict!its!motion!to!maintain!coplanarity!throughout!multiple!rings!
composing!the!conjugated!system.!!Hydrogen!bond!networks!have!been!identified!in!
the!crystal!structures!of!other!RFPs!including!mNeptune,!mRouge,!eqFP650,!
eqFP670,!mCherry,!TagRFP675,!and!mRojoA!and!may!play!a!critical!role!in!tuning!
the!emission!(absorption)!wavelength!through!excited!(ground)!state!
stabilization11V12,!47V50.!!Moreover,!nonradiative!deactivation!of!the!excited!
population!resulting!from!enhanced!flexibility!about!the!extended!conjugated!
system!might!explain!their!comparably!low!quantum!yields!relative!to!bluerV
emitting!species.!!
1.2!Nonlinear(Femtosecond(Spectroscopy(
Evolution#of#Time;Resolved#Experimentation#
This!section!will!briefly!survey!the!evolution!of!timeVresolved!methods!as!
they!developed!coincident!with!advances!in!detection!and!light!source!technology.!!
The!seminal!accounts!of!timeVresolved!spectroscopy!centered!on!nonreactive!
solvation!dynamics!of!simple!organic!dyes!in!solution.!!Pioneering!works!of!
Bakshiev!and!Ware!utilized!fluorescence!upconversion!spectroscopy!with!streak!
cameras!to!decipher!nanosecond!responses!of!solvated!dyes51V53.!!This!work!
spawned!an!entire!field!of!research!into!addressing!the!molecular!movements!about!
an!excited!optical!probe54.!
These!methods!were!also!applied!to!biological!systems!where!it!was!
discovered!that!proteins!demonstrated!a!response!unique!from!a!probe!in!bulk!

!8!
solvent55V57.!!Early!experiments!revealed!relaxation!across!a!broader!time!range!
compared!to!dyes!in!simple!solvents58.!!The!protein!response!was!also!found!to!be!
highly!nonexponential,!characteristic!of!inherent!heterogeneity!that!was!attributed!
to!natural!multiplicity!of!conformational!substates.!!These!observations!are!
consistent!with!the!energy!landscape!picture,!the!predominant!physical!model!that!
connects!protein!dynamics!to!their!function59V60.!
The!stage!was!set!to!connect!these!phenomena!with!protein!function!itself.!!
However,!with!particular!regard!to!a!complex!energy!landscape,!probing!specific!
aspects!of!required!novel!strategies!to!improve!experimental!clarity.!!Methods!
including!photoinitation!via!flash!photolysis,!fluorescence!energy!transfer!or!excited!
state!quenching!to!measure!spatial!proximity,!excited!state!proton!transfer,!and!
incorporation!of!exogenous!probes!were!used!to!better!visualize!a!particular!
reaction61V64.!!These!preliminary!investigations!spawned!investigation!of!more!
complex!ideas!such!as!conformational!heterogeneity,!allostery,!and!further!
mitigating!the!effect!of!ensemble!averaging,!which!motivated!the!development!of!
more!sophisticated!experimentation65V67.!!
Modern!biophysical!spectroscopic!applications!have!revealed!a!broad!range!
of!photochemical!reactions.!!A!few!examples!include!understanding!the!
photoisomerization!in!the!photoreceptor!rhodopsin!that!constitute!the!primary!
steps!in!human!vision,!exciton!transfer!dynamics!in!photosynthetic!lightVharvesting!
complexes,!protein!folding!dynamics,!DNA!damage!repair!mechanisms,!and!redox!
chemistry!of!several!enzymes32,!68V73.!!Other!efforts!have!focused!on!more!

!9!
fundamental!facets!governing!protein!motions!in!canonical!systems!(e.g.!COVligand!
dissociation!dynamics!in!myoglobin)!to!help!develop!a!unified!model!of!protein!
structureVfunction!relationships74V75.!!However,!while!the!field!has!greatly!evolved!
since!the!infancy!of!spectroscopic!analysis,!modern!informationVrich!techniques!
offer!insight!into!a!host!of!new!biophysical!dilemmas!including!investigation!of!
correlated!motions!and!longVrange!allosteric!interactions!on!timescales!previously!
inaccessible!to!earlier!methods.!!!!!
The!invention!of!ultrashort!light!sources!has!accelerated!the!evolution!of!
timeVresolved!spectroscopy76V77.!!In!particular,!modern!femtosecond!lasers!produce!
pulse!durations!on!the!order!of!molecular!vibrations!and!enable!investigation!of!the!
fastest!motions!in!condensed!phase!systems.!!Such!methodology!is!well!suited!to!
unravel!a!wide!spectrum!of!protein!motions!by!depositing!energy!instantaneously!
and!in!discrete!quantities!leaving!the!excited!probe!out!of!equilibrium!with!the!
surrounding!environment.!!The!time!dependent!response!an!excited!probe!is!most!
generally!represented!as!a!time!correlation!function!of!the!electronic!energy!gap!!
C (t ) ∝ δω10 (t ) δω10 ( 0 )

!

!

!

!

!
(1.1)

where!δω10#is!the!energy!gap!of!a!ground!to!first!excitedVstate!electronic!transition.!!
Monitoring!this!quantity!over!timescales!characteristic!of!molecular!relaxation!and!
subsequent!Fourier!transformation!yields!a!vibrational!spectral!density!(ρ(ω))!
encompassing!the!spectrum!of!motions!participating!in!systemVbath!relaxation78!

λ=1 ∞∫ ωρ (ω ) dω
π 0
!

!

!

!

!

(1.2)!

10!
!
where!λ!is!the!total!nuclear!reorganization!energy.!!This!is!perhaps!the!most!critical!
variable!accessed!by!transient!spectroscopic!experimentation.!!Femtosecond!
spectroscopic!techniques!have!evolved!to!encompass!spectral!regions!from!the!midV
infrared!to!xVrays79V83.!!These!experiments!yield!systemVbath!coupling!over!a!wide!
time!range!with!varying!sensitivity.!!Their!resolving!power!is!owed!to!both!
experimental!sophistication!and!the!nature!of!the!spectroscopic!probe.!!The!
practical!distinctions!between!femtosecond!and!conventional!spectroscopy!can!be!
viewed!from!the!perspective!of!this!sensitivity,!which!varies!widely!in!both!time!and!
length!scales.!!!A!discussion!of!the!theoretical!underpinnings!of!nonlinear!
spectroscopy!will!now!follow.!
Response#Function#Formalism#
As!referenced!by!name,!nonlinear!spectroscopic!methods!exploit!lightVmatter!
interactions!beyond!a!linear!response.!!LightVmatter!interactions!are!often!
characterized!by!an!induced!polarization,!expressed!below!as!a!Taylor!Series!
expansion!with!respect!to!the!electric!field78.!
P (t ) = P ( ) + P ( ) + P ( ) + P ( ) + ⋅⋅⋅

!

!

!

!
(1.3)

P = χ ( ) E + χ ( ) EE + χ ( ) EEE +... !

!

!

(1.4)!

0

1

2

3

or!
1

2

3

Here,!χ(n)!represents!the!nth!order!nonlinear!susceptibility!tensor,!E!is!the!incident!
electric!field,!and!P#is!the!induced!polarization.!!The!susceptibility!tensor!arises!from!
symmetry!considerations!within!the!nonlinear!media!and!serves!as!a!useful!
selection!rule!for!practical!application!of!various!nonlinear!techniques.!!For!example,!

11!
!
χ(2)!processes!such!as!Second!Harmonic!Generation!(SHG)!and!Sum!Frequency!
Generation!(SFG)!are!possible!only!in!noncentrosymmetric!media!lacking!an!
inversion!center84.!!The!experiments!conducted!in!this!project!utilize!femtosecond!
light!sources!with!sufficient!peak!intensities!to!access!the!third!order!response!of!
various!biological!probes.!!!
TimeVdependent!quantum!mechanics!is!required!to!adequately!describe!
lightVmatter!interactions!and!their!associated!dynamic!evolution!in!the!condensed!
phase.!!This!characterization!relies!on!the!density!matrix!operator,!!,"used"to"treat"systems"of"statistically"mixed"states85."""

"

"

"

"

"

"

ρnm = n ρ n = an " """(1.5)"

ρnm = n ρ m = an am* " """(1.6)"

2"

Diagonal!terms!represent!population!states,!while!offVdiagonal!terms!are!coherence!
between!two!states,!leading!to!population!relaxation!and!dephasing!(decoherence)!
respectively.!!The!time!evolution!of!these!Eigenstates!follows!from!the!quantum!
Liouville!equation!

!

!

!

!

!

!

!

!

!

!

∂ρ −i
= [ H, ρ ] !
∂t !

!

!

!

(1.7)! !

! = !! + ! ! !

!

!

!

(1.8)!

Here,!H!is#the!total!molecular!Hamiltonian,!H0!is!the!unperturbed!Hamiltonian!and!
V(t)!is!the!perturbation!describing!the!lightVmatter!interaction.!!If!considering!only!
electricVdipole!transitions!the!final!term!becomes!

12!
!
!

!

!

!

!

! ! = −! ∙ !!!

!

!

!

(1.9)!

where!µ!is!the!electronic!dipole!moment!and!E!is!the!incident!electric!field.!!
Nonlinear!polarizations!are!most!easily!treated!as!higher!order!perturbations!of!the!
density!matrix.!!The!time!dependent!perturbed!density!matrix!can!be!evaluated!by!
inserting!into!Equation!1.7!
2tt" i% tnρ ( ) (t ) = $ − ' ∫ −∞ dtn ∫ −∞ dtn−1... ∫ −∞ dtt *+V (tn ), *+V (tn−1 ), *+..., *+V (t1 ), ρeq ,-...,-,-,- ! (1.10)!
# !&The!total!nonlinear!polarization!is!generally!expressed!as!
P ( ) (t ) = µρ ( ) (t ) !
n

n

!

!

!

(1.11)!

After!organizing!terms,!this!leads!to!a!solution!for!the!polarization!separated!among!
several!unique!response!functions.!!
∞

∞

0

0

P ( ) (t ) = ∫ d τ n ⋅⋅⋅ ∫ d τ 1R( ) (τ 1, τ 2 ,...τ n ) E1 (t − τ n − ⋅⋅⋅ − τ 1 ) ⋅ ⋅⋅En (t − τ n )
n

n

!

!
(1.12)

Here,!R(n)!is!the!nth!order!nonlinear!response!function!
n!i$
R (τ 1, τ 2 ,...τ n ) = # & θ (τ 1 )θ (τ 2 )...θ (τ n ) ×
"!%
(n)

{

Tr )*)*... )*µ (τ n + τ n−1 +...τ 1 ), µ (τ n−1 + τ n +...τ 1 )+,,...+, µ ( 0 )+, ρeq

}

!!

!
(1.13)

A!total!of!2n!terms!are!possible!stemming!from!multiple!interactions!with!the!bra!
and!ket!side!of!the!density!matrix.!!However,!after!accounting!for!all!possible!
interactions,!a!total!of!48!terms!exist!for!a!twoVlevel!third!order!response.!!!
A!few!notable!experimental!tools!are!utilized!to!simplify!the!number!of!
unique!pathways.!!First,!phaseVmatching!enables!spatial!isolation!of!specific!

13!
!
nonlinear!responses.!!Early!applications!of!NMR!spectroscopy!were!carried!out!in!a!
collinear!geometry,!which!necessitated!separation!of!excitation!pulses!with!the!
emitted!signal!field.!!Conventional!nonlinear!optical!experiments!typically!utilize!a!
noncollinear!excitation!geometry86.!!The!most!commonly!used!is!the!BOXCARs!
geometry,!stemming!from!Coherent!Antistokes!Raman!Spectroscopy!(CARS),!where!
the!excitation!beams!are!incident!on!the!corners!of!a!square.!!This!configuration!
enables!spatial!isolation!of!the!emitted!third!order!polarization!and!ideal!phaseVmatching!efficiency!considering!the!symmetric!arrangement!of!the!beams.!!Second,!
reordering!the!relative!arrival!time!of!excitation!pulses!at!the!sample!position!
within!the!BOXCARs!geometry!enables!selective!sampling!of!different!nonlinear!
responses!without!physical!realignment!of!excitation!beams.!!Finally,!diagrammatic!
methods!are!a!useful!tool!for!organizing!and!interpreting!nonlinear!response!
functions78.!!In!particular,!doubleVsided!Feynman!diagrams!describe!transitions!
within!the!density!matrix!with!a!specific!time!ordering!and!phaseVmatching.!!A!brief!
primer!of!this!method!is!given!in!Figure!1.3.!!
!

14!
!

Anatomy of a Feynman Diagram
Time•Vertical lines represent time evolutation of bra and ketsides of density matrix

aa

t3

ba

•Time increases from bottom up

t2

aa
ab
aa

•Light-matter interactions are indicated by solid arrows

t1•Inward and outward-going arrows represent photon
absorption and emission respectively•Emitted signal is signified by a dashed arrow•The system must begin and end in a population state!
Figure(1.3.((Guidelines!for!interpreting!doubleVsided!Feynman!diagrams!used!to!
described!nonlinear!lightVmatter!interactions.!!(
The!diagrams!in!Figure!1.4!describe!the!most!commonly!encountered!pathways!for!
electronic!spectroscopy!of!threeVlevel!molecular!systems.!

15!
!
Rephasingksig=-k1+k2+k3

R1

R2

R3

00

00

11

10

10

21

11
01
00

00

11

01
00

01
00

Nonrephasing

ksig=k1-k2+k3

R4

R5

R6

00

00

11

10

10

21

00
10
00

11

11
10
00

10
00

Double Quantum Coherence
R7

R8

00

11

10

21

20
10
00

20

ksig=k1+k2-k3

10
00

!
Figure(1.4.!!Third!order!nonlinear!optical!responses!for!a!threeVlevel!system.!!These!
can!be!categorized!in!terms!of!phaseVmatching!conditions!as!rephasing,!
nonrephasing,!or!double!quantum!coherence!contributions.!
Bath#Models#
The!countless!permutations!of!sidechain!flexibility,!conformational!
heterogeneity,!hydrogen!bonding,!and!innumerable!tertiary!interactions!in!
biological!systems!form!the!complex!energetic!landscape!introduced!above.!!

16!
!
Unraveling!these!seemingly!innumerable!variables!represents!a!central!challenge!
for!deciphering!biophysical!functional!mechanisms.!!In!the!context!of!electronic!
spectroscopy,!it!explains!the!typically!congested!absorption!spectra!of!biophysical!
probes!that!complicate!physical!interpretation.!!Electronic!transition!frequencies!
are!modulated!by!coupling!with!nuclear!degrees!of!freedom!of!the!chromophore!
itself!and!that!of!the!surrounding!bath.!!These!interactions!are!too!complex!for!a!
complete!analytical!treatment!and!thus,!require!some!degree!of!approximation!to!
accurately!predict!experimental!data.!!This!section!will!describe!the!general!
foundations!concerning!systemVbath!interactions!and!discuss!the!prominent!bath!
models!utilized!in!theoretical!treatment!of!electronic!absorption!lineshapes.!!!!!!!!

1

10

hv
0

(t )
i
10

(t ) =

10

+ i+

10

(t )

Homogeneous
Inhomogeneous!

Figure(1.5.((The!time!evolution!of!an!electronic!transition!frequency!in!the!
condensed!phase!can!be!dissected!into!separate!homogeneous!and!inhomogeneous!
components.!
The!electronic!absorption!lineshape!(σ(ω))!can!be!generally!expressed!as!a!
Fourier!transform!of!the!complex!lineshape!function!g(t)!containing!all!systemVbath!
interaction!information.!!!

17!
!

σ (ω ) =1 ∞iω t−g t
dte−iωt e 10 ( ) ! !
∫
2π −∞

!

!

(1.14)!

In!its!simplest!form,!the!lineshape!function!is!treated!as!linear!damping!term!(Γ),!
known!as!the!Bloch!limit,!and!is!characterized!by!a!Lorentzian!absorption!
lineshape87.!!This!model!assumes!an!equal!(homogeneous)!treatment!for!all!
members!of!the!ensemble.!!In!the!opposite!limit,!g(t)!is!assumed!to!be!a!quadratic!
term!(Δ2)!and!leads!to!a!Gaussian!absorption!lineshape.!!This!model!is!frequently!
used!to!represent!cases!of!large!inhomogeneous!broadening!where!the!system!
exists!of!independent!absorbers!having!a!statistically!normal!distribution!of!
transition!frequencies!within!the!Central!Limit!Theorem.!

a

b

~2"

! egi ( 0(0))
i10! egi ((tt) )i

10!!10eg"!!10eg"Figure(1.6.!!(a)!Inhomogeneous!Gaussian!absorption!lineshape!with!underlying!
Lorentzian!subensembles.!(b)!Time!dependent!transition!frequency!of!a!single!
excited!oscillator.!!
A!stochastic!model,!originally!introduced!by!Kubo!et#al.,!incorporates!
continuous!interpolation!between!fast!and!slow!components!of!bath!fluctuation88.!!
This!handling!was!sufficient!for!description!of!resonance!Raman!and!fluorescence!
processes,!but!is!inadequate!in!cases!involving!large!changes!in!electron!density!or!

!

18!
!
solvent/solute!reorganization!along!a!particular!reaction!coordinate89.!!A!stochastic!
bath!is!adequate!for!probing!general!aspects!of!absorption!features,!however,!a!
more!complete!model!is!necessary!to!obtain!accurate!absorptive!lineshapes!and!
their!transient!behavior!as!is!typically!sought!by!nonlinear!spectroscopy.!!The!
current!standard!theoretical!treatment!is!the!multimode!Brownian!oscillator78.!!This!
model!assumes!the!solute!is!subject!to!a!quantum!mechanical!bath!subject!to!
LangevinVlike!friction.!!It!converges!to!the!stochastic!results!within!classical!limits,!
but!with!proper!inclusion!of!Stokes!shift!and!nonVMarkovian!dynamics.!!Its!
characteristic!lineshape!function!at!high!temperature!is!generalized!as!

# t
g (t ) = ∑%iλ j ∫ dt1M j (t1 ) + Δ 2j
0
j $

t1
& Δ2 t 2
dt
dt
Mt∫ 1 ∫ 2 j ( 2 )(' + in2 !!! !
00t(1.15)!

where!the!total!reorganization!energy,!λj,!is!partitioned!among!all!participating!
solvent!modes!with!weighting!Δj.!
1.3(Survey(of(Nonlinear(Optical(Methods(
Regardless!of!the!precise!bath!treatment,!electronic!lineshapes!of!
biomolecules!are!often!broad!and!featureless!enhancing!the!difficulty!of!interpreting!
underlying!structure!or!dynamics.!!Extracting!this!information!is!facilitated!by!the!
experiments!described!in!this!section.!!A!survey!of!experimental!methods!employed!
in!this!thesis!is!given!below.!!Precise!data!collection!procedures!will!be!presented!in!
a!later!chapter.!!!
!
!

19!
!
Transient#Absorption#Spectroscopy#Tksig=2kpu-kpr

Time!
Figure(1.7.((Beam!geometry!and!pulse!sequence!for!a!transient!absorption!
spectroscopy!experiment.!!
Transient!absorption!(TA)!spectroscopy!is!conducted!by!measuring!
differential!intensity!of!the!probe!field!with!and!without!exposure!of!a!preceding!
pump!field.!!It!is!treated!as!a!3rd!order!interaction!with!a!2kpuVkpr(phase!matching!
condition.!!The!detected!signal!intensity!can!be!expressed!as90!

ΔI (T ) =22nc
E pr' Esig (T ) − E pr'
!
4π

{

} !

!

(1.16)!

where!n!is!the!refractive!index,!c!is!the!speed!of!light,!and!Epr!and!Esig!are!the!probe!
and!nonlinear!signal!fields!respectively.!!In#situ!removal!of!Epr!by!chopping!the!pump!
beam!leaves!only!the!cross!term!and!following!substitution!for!Esig!yields!
3'
ΔI (T ) = 2ω sigl Im "# E pr
P ( ) (T )$% !

!

!

(1.17)!

where!Im(P(3))!represents!the!imaginary!(absorptive)!part!of!the!3rd!order!
polarization.!!Given!this!relationship,!the!TA!response!is!sensitive!to!both!ground!
and!excited!state!dynamics.!!Measurements!are!commonly!spectrallyVresolved!to!

20!
!
provide!simultaneous!dynamic!information!across!a!wide!spectral!window.!!Since!
TA!is!not!inherently!backgroundVfree,!sophisticated!detection!schemes!such!as!lockV
in!amplification!have!helped!to!improve!experimental!sensitivity.!!
Transient#Grating#Spectroscopy##
A!threeVpulse!analogue!of!the!transient!absorption!technique!is!transient!
grating!(TG)!spectroscopy.!!The!temporal!overlap!of!k1!and!k2!(τ=0)!imprints!a!
spatial!interference!on!the!irradiated!sample!volume!leading!to!a!periodic!area!of!
high!and!low!intensity.!!This!is!commonly!described!as!a!population!grating!and!its!
time!evolution!is!sensitive!to!excited!state!lifetime,!spectral!diffusion,!and!solvation!
dynamics91V92.!!The!TG!signal!contains!both!ground!and!excited!state!components!
stemming!described!by!the!response!functions!below.!!Like!TA!spectroscopy,!
spectrallyVresolved!detection!enables!quantification!of!solvation!dynamics.!!
However,!TG!spectroscopy!is!lessVfrequently!used!likely!due!to!the!need!for!a!third!
excitation!beam.!

Tksig=-k1+k2+k3

Time"
Figure(1.8.((Beam!geometry!and!pulse!sequence!for!a!typical!transient!grating!
spectroscopy!experiment.!

21!
!

RSE

RGSB

RESA

00

00

11

10

10

21

11
01
00

00
01
00

11
01
00
"

Figure!1.9.!!Third!order!lightVmatter!interactions!for!a!transient!grating!experiment!
of!a!threeVlevel!system.!!!
Photon#Echo#Spectroscopy#
As!indicated!above,!the!ensemble!average!is!only!partially!subverted!with!
femtosecond!time!resolution.!!The!highly!coupled!structural!arrangement!of!
biomolecules!yields!complex!absorption!spectra!with!several!overlapping!features.!!
Moreover,!these!features!may!be!highly!separable!in!the!time!domain!rendering!
them!indecipherable!by!conventional!methods.!!These!divisions!in!time!are!known!
as!the!homogeneous!(fast)!and!inhomogeneous!(slow)!limits!and!can!be!described!
by!their!respective!linewidths!as!illustrated!in!Figure!1.6.!!
Photon!echo!techniques!exploit!the!inherent!inhomogeneity!of!chemical!
resonances!in!the!condensed!phase!and!their!molecular!dephasing!and!rephasing!
processes.!!These!methods!were!first!applied!to!NMR!spectroscopy!following!the!
discovery!of!spin!echoes!and!are!the!subject!of!several!extensive!reviews93.!!NMR!
correlation!spectroscopy!is!used!extensively!in!the!pursuit!of!protein!structure!and!
measurement!of!slow!(µsVms)!dynamics94V95.!!Modern!ultrafast!lasers!have!led!to!
analogues!applied!at!infrared,!visible,!ultraviolet!wavelengths96V98.!!These!

22!
!
experiments!enable!a!similar!examination!of!buried!vibrational!and!electronic!
probes!in!biological!systems,!albeit!at!different!time!and!length!scales.!!
Photon!echo!spectroscopy!relies!on!the!spontaneous!collective!rephasing!of!
independent!dipoles99.!!This!phenomenon!is!considered!the!optical!analogue!of!a!
spin!echo!famously!introduced!by!Hahn93,!100.!!In!a!molecular!context,!optical!photon!
echoes!represent!the!collective!dephasing!and!subsequent!rephasing!of!independent!
dipoles.!!In!a!condensed!phase!system,!the!dephasing/rephasing!process!is!driven!
by!coherent!electronic!excitation!with!an!ultrashort!light!source!combined!with!the!
intrinsic!heterogeneity!of!transition!frequencies!as!described!above.!!!!!

Ѯ0Dephasing

Population

Rephasing!

Figure(1.10.((Evolution!of!the!collective!phase!of!the!excited!volume!throughout!a!
photon!echo!spectroscopy!experiment.!!!
The!evolution!of!the!collective!phase!upon!coherent!excitation!of!the!excited!volume#
is!illustrated!in!Figure!1.10!and!can!be!expressed!as!!!
iφi (t ) ∝ eiω10t ! !

!

!

!

(1.18)!

23!
!
Here,!the!extent!of!sample!dephasing!or!rephasing!is!modulated!by!intrinsic!
heterogeneity!together!with!spectral!diffusion!occurring!throughout!the!population!
time!interval100.!!As!such,!dynamic!information!is!preserved!over!a!time!interval!
spanning!femtoseconds!to!several!seconds!unobscured!by!sample!heterogeneity.!!
This!criterion!renders!photon!echo!spectroscopy!intrinsically!more!powerful!than!
the!TA!and!TG!methods!described!above.!!!!!

τ

T

t

LO

ksig=-k1+k2+k3

Time!
Figure(1.11.((Beam!geometry!and!pulse!sequence!for!a!heterodyneVdetected!
stimulated!photon!echo!spectroscopy!experiment.!
Figure!1.11!illustrates!the!BOXCARs!beam!geometry!and!pulse!sequence!used!
to!generate!a!photon!echo!signal.!!Scanning!through!the!τ!dimension!yields!an!
approximate!Gaussian!distribution!of!signal!intensity!and!its!peak!position!in!time!is!
known!as!the!“peak!shift”.!!This!value!plotted!over!a!series!of!popuation!times!is!
proportional!to!the!energy!gap!time!correlation!function!and!forms!the!basis!of!
Three!Pulse!Photon!Echo!Peak!Shift!(3PEPS)!spectroscopy.!!Moreover,!asymptotic!
peak!shift!values!also!relate!to!the!homogeneous!(Γ)!and!inhomogeneous!(Δin)!

24!
!
linewidths!expressed!below!in!the!limit!of!a!two!level!system!coupled!to!a!harmonic!
bath!with!impulsive!excitation101.!

τ * (T = 0) ≈ ( Γπ )

−1

!

!

τ * (T → ∞) ≈ Δ in2 ( Γ + Δ in2 )!

!

!
(1.19)! !

!

(1.20)!

−1

Multidimensional#Spectroscopy#
Photon!echo!phenomena!can!also!be!extended!with!a!second!frequency!
dimension!where!a!resonance!is!spread!across!multiple!axes.!!This!treatment!yields!
a!transient!correlation!map!of!excitation!and!emission!frequencies!about!a!fixed!
population!time!and!captures!spectral!diffusion,!coherent!dynamics,!and!energy!
transfer!events!within!the!ensemble.!!Generally!known!as!multidimensional!
spectroscopy,!this!strategy!has!been!applied!to!nuclear!spin,!vibrational!and!
electronic!transitions!and!is!especially!valuable!in!congested!spectral!regions!typical!
of!condensed!phase!systems102V105.!!However,!the!execution!of!proper!spectral!
interferometry!complicates!multidimensional!techniques.!!This!becomes!
exceedingly!challenging!when!fluctuations!of!the!surrounding!environment!cause!
appreciable!phase!drifts!at!optical!frequencies.!!Several!strategies!involving!both!
passive!and!active!phase!stabilization!have!been!proposed106V109.!!!
A!general!description!of!typical!multidimensional!spectra!reveals!its!
resolving!power.!!First,!Fourier!transformation!with!respect!to!the!τ!and!t!time!
delays!yields!a!twoVdimensional!frequency!plot!correlating!excitation!(initial)!and!
emission!(final)!frequencies!of!a!given!oscillator110V111.!!Next,!data!are!collected!for!a!
series!of!population!times!enabling!investigation!of!transient!spectral!dynamics.!!

25!
!
The!figure!below!illustrates!expected!spectral!features!for!a!condensed!phase!
response.!!Elongation!along!the!diagonal!at!early!T!indicates!nonVMarkovian!
behavior!consistent!with!an!inhomogeneously!broadened!transition112.!!While!at!
long!population!time,!the!spectrum!appears!more!symmetric!as!the!system!
eventually!samples!its!entire!frequency!space.!!!

Ωexc=Ωemis

Early T

Ωexc=Ωemis

Late T

~Γ

Ωemis

Ωemis
~Δ

Ωexc

Ωexc

Figure(1.12.!!Expected!twoVdimensional!spectral!evolution!of!a!twoVlevel!electronic!
system!in!the!condensed!phase.!!(a)!Inhomogeneous!broadening!is!manifest!at!early!
population!time!as!elongation!along!the!diagonal.!(b)!A!symmetric!peak!is!predicted!
at!population!times!beyond!the!characteristic!bath!correlation!time.!
The!case!of!population!exchange!is!presented!in!Figure!1.13.!!At!early!
population!time,!two!peaks!on!the!diagonal!appear!representing!contributions!from!
either!state!with!unique!transition!energies.!!At!later!population!time,!cross!peaks!
above!and!below!the!diagonal!appear!representing!population!transfer!from!the!
highVtoVlow!and!lowVtoVhigh!energy!states!respectively.!!This!situation!is!relevant!
for!systems!having!closely!spaced!electronic!modes!such!as!exciton!migration!in!

!

26!
!
photosynthetic!light!harvesting!complexes!and!isomerization!events!in!biological!
systems69,!113.!

Ωexc=Ωemis

Early T

Ωexc=Ωemis

Late T

Ωemis

Ωemis

Ωexc

Ωexc

!

Figure(1.13.!!Expected!twoVdimensional!spectral!evolution!for!the!case!of!electronic!
population!transfer.!!(a)!Two!peaks!fall!along!the!diagonal!at!early!times!
representing!the!independent!transitions!of!each!mode.!(b)!Cross!peaks!appear!at!
later!population!times!that!signify!population!transfer!between!the!low!and!high!
energy!modes.!
(1.4(Thesis(Objectives(
The!above!accounts!lay!the!foundation!for!investigation!of!RFP!photophysical!
properties!with!femtosecond!nonlinear!spectroscopy.!!Previous!exploration!of!these!
phenomena!has!been!largely!restricted!to!the!standard!methods!of!structural!
biology,!which!are!incapable!of!resolving!the!complete!range!of!molecular!motions!
thought!responsible!for!their!complex!behavior.!!TimeVresolved!electronic!
spectroscopy!is!uniquely!suited!to!quantify!the!timescales!of!local!chromophoreV
sidechain!interactions.!!However,!few!previous!accounts!have!addressed!the!
dynamic!behavior!of!RFPs!including!the!molecular!origin!of!their!red!emission.!!This!

27!
!
thesis!aims!to!make!these!connections!and!help!establish!nonlinear!femtosecond!
spectroscopy!as!a!versatile!tool!for!biophysical!research.!!Specific!objectives!include!
investigating!the!hydrogen!bond!network!in!mPlum,!one!of!the!farthest!known!redV
emitting!RFPs.!!Transient!grating!spectroscopy!is!used!to!probe!specific!
chromophoreVsidechain!interactions!thought!responsible!for!its!extended!Stokes!
shift.!!These!results,!corroborated!with!MD!simulations,!reveal!hydrogen!bond!
interconversion!unique!to!the!reddestVemitting!members!among!a!panel!of!strategic!
point!mutants.!!The!principles!acquired!in!this!study!were!next!applied!to!
investigate!a!similar!hydrogen!bond!network!in!TagRFP675,!the!farthest!redV
emitting!RFP!discovered!to!date.!!Finally,!multidimensional!spectroscopy!was!
employed!to!characterize!the!excited!state!electronic!structure!of!mPlum.!!This!
information!is!potentially!useful!for!understanding!the!dark!state!conversion!
processes!that!hinder!full!utilization!of!RFPs!in!advanced!imaging!applications.!
!
!
!
!
!
!
!
!
!
!
!

28!
!
Chapter(II(Experimental(Methods(!
2.1(Description(of(Light(Source!
The!physical!interpretations!of!this!thesis!were!derived!from!measurement!
of!molecular!dynamics!extracted!from!nonlinear!spectroscopic!experimentation.!!
From!a!macroscopic!perspective,!these!dynamics!are!conveniently!treated!as!a!time!
correlation!function!of!the!electronic!energy!gapVa!common!model!to!describe!a!
thirdVorder!response78.!!To!facilitate!these!measurements,!a!light!source!capable!of!
generating!femtosecond!laser!pulses!resonant!with!an!electronic!transition!of!
interest!with!sufficient!bandwidth!and!intensity!is!required.!!The!sections!that!
follow!describe!hardware!and!device!development!to!facilitate!this!experimentation.!!
First,!the!light!source!used!to!carry!out!spectroscopic!experimentation!will!be!
described.!!Next,!aspects!of!pulse!dispersion!compensation!including!attempts!at!
adaptive!pulse!shaping!are!presented.!!Finally,!specific!details!concerning!
experimental!data!acquisition!procedures!and!the!preparation!of!biological!samples!
will!be!discussed.!
A!single!light!source!was!used!for!all!timeVresolved!experimentation.!!This!
consisted!of!a!Noncollinear!Optical!Parametric!Amplifier!(NOPA)!pumped!by!a!
Ti:Sapphire!regenerative!amplifier!diagrammed!below!in!Figure!2.1.!!Generation!of!
ultrashort!laser!pulses!takes!places!in!multiple!steps!and!the!individual!elements!are!
outlined!below.!

29!
!

Opus

KML Ti:Sapphire
Oscillator: 800 nm, 76 MHz

Photonics Industries
DH20

Regenerative Amplifier:
800 nm, 20 KHz~150 fs

NOPA: 450-700
nm, 20 KHz~15 fsPulse compressor

JILA MONSTR

[

]

3

!
Figure(2.1.((Outline!of!the!light!source!used!for!timeVresolved!experimentation!
beginning!with!generation!of!ultrafast!laser!pulses!at!visible!wavelengths!and!
ending!compressed!to!their!shortest!duration!at!the!sample!position.!!!
Commercial#Amplifier#System#
The!principal!light!source!is!a!commercial!Ti:Sapphire!amplifier!system!
manufactured!by!KM!Labs!(WyvernV20).!!This!system!outputs!an!800!nm!pulse!train!
with!tunable!repetition!rate!(10V100!kHz)!and!a!compressed!duration!of!~50!fs.!!
Modern!stateVofVtheVart!turnkey!amplifiers!exploit!a!few!prevalent!technologies!that!
have!accelerated!the!development!of!ultrafast!spectroscopies!in!recent!years.!!!!
Solid#State#Gain#Media##
TitaniumVdoped!sapphire!(Ti:sapphire)!has!effectively!supplanted!laser!dyes!
as!the!standard!gain!medium!used!in!femtosecond!amplifier!systems.!!Its!utility!is!
owed!to!several!favorable!properties,!namely,!a!large!gain!bandwidth!of!700V1100!
nm!permitting!tunable!ultrashort!pulse!generation!and!high!thermal!conductivity!
and!energy!storage!density!(~1!J/cm2)114V115.!!Such!conditions!are!a!great!
improvement!over!the!tedious!operation!of!dye!lasers!requiring!continual!
maintenance!of!hazardous!substances!with!inferior!performance.!!!
#

30!
!
Passive#Modelocking#Modelocking!requires!the!matching!of!phase!for!several!longitudinal!modes!
within!a!laser!cavity116.!!The!result!is!a!pulse!train!with!frequency!modulated!by!the!
cavity!length!and!duration!inversely!dependent!on!the!number!of!locked!
longitudinal!modes.!!Several!modelocking!schemes!have!been!devised!and!are!
categorized!as!either!active!or!passive.!!The!former!involves!introduction!of!electroV!
or!acoustoVoptic!modulation!to!achieve!periodic!intracavity!loss,!but!are!generally!
limited!to!picosecond!pulse!durations!given!their!instrinsically!slow!response!and!
typically!require!complex!electronic!hardware.!!Passive!locking!strategies!typically!
rely!on!a!static!optical!component,!such!as!an!intracavity!saturable!absorber!(e.g.!
SESAM)!to!achieve!momentary!and!predictable!resonator!loss117.!!A!much!more!
rapid!modulation!is!possible!compared!to!active!modelocking!enabling!femtosecond!
pulse!durations!making!it!suitable!for!ultrashort!light!sources.!!!
Chirped#Pulse#Amplification#
Most!modern!ultrafast!nonlinear!spectroscopic!applications!require!pulse!
energies!beyond!that!possible!with!CW!pumped!amplifier!systems!at!medium!
repetition!rates!(10V100!kHz)118.!!As!such,!current!stateVofVtheVart!systems!rely!on!
pulsed!sources!to!seed!the!amplification!stage.!!Chirped!Pulse!Amplification!(CPA)!
has!been!adopted!as!a!principal!strategy!to!achieve!much!higher!pulse!energies!
while!circumventing!the!natural!physical!limits!of!gain!media!when!exposed!to!the!
high!peak!powers!of!ultrafast!seed!pulses119.!!This!strategy!has!led!to!an!order!of!
magnitude!increase!in!pulse!energies!and!is!typical!of!modern!ultrafast!light!sources.!!!

31!
!
In!this!system,!a!reflective!grism!pair!as!illustrated!in!Figure!2.2!induces!
temporal!chirp!on!the!amplifier!seed!beam.!!Grisms!combine!the!dispersive!
properties!of!both!gratings!and!prisms!and!provide!an!efficient!means!of!
introducing!seed!dispersion!in!broadband!systems120.!!A!grism!provides!a!few!
advantages!over!the!standard!grating!stretcher!method.!!First,!its!design!is!premised!
on!the!nearly!constant!GVD/TOD!ratio!in!most!materials!and!introduces!negative!
GVD!and!TOD!as!a!function!of!their!separation.!!As!such,!grism!systems!represent!an!
example!of!DownVChirped!Pulse!Amplification!(DCPA)!and!compression!is!achieved!
by!simply!transmitting!the!amplified!beam!through!material!of!the!proper!GVD/TOD!
ratio121.!!This!avoids!the!cumbersome!task!of!using!a!combination!of!prisms!and!
gratings!to!carry!out!final!pulse!compression,!which!suffer!from!lowVthroughput!and!
require!tedious!alignment.!!Finally,!a!grism!stretcher!avoids!the!wellVknown!
limitations!multiVpass!grating!stretchers,!e.g.!alignment!challenges!and!low!
throughput.!!The!only!adjustable!parameters!in!our!system!are!the!separation,!d,!
and!incidence!angle!controlled!by!a!micrometer!rotation!stage.!!!

32!
!

G1
RM1

d

G2

To regenerative
amplifier
800 nm fromoscillator, 80 MHz!
Figure(2.2.((Grism!pair!stretcher!used!to!chirp!oscillator!output!seeding!WyvernV20!
regenerative!amplifier.!

Compressor

Ti:Sapphire

Q Switch

532 nm CW Pump

Oscillator(
Figure(2.3.((Beam!diagram!of!the!KM!Labs!WyvernV20!regenerative!amplifier!light!
source.!
Effective!timeVresolved!spectroscopic!experimentation!demands!both!precise!
time!resolution!as!well!as!a!tunable!light!source!capable!of!exploring!a!wide!range!of!
electronic!transitions.!!Optical!Parametric!Amplification!(OPA)!represents!the!most!
reliable!method!to!produce!ultrashort!optical!pulses!satisfying!these!conditions.!!

33!
!
This!phenomenon!is!a!χ(2)!process!that!occurs!in!nonlinear!media!lacking!inversion!
symmetry.!!Most!commonly,!βVbarium!borate!(BBO)!is!used!due!to!its!high!damage!
threshold,!low!group!velocity!dispersion,!and!broad!transparency!range122V123.!!In!
this!process,!a!high!frequency,!high!intensity!beam!(pump)!amplifies!a!lower!
frequency,!lower!intensity!beam!(signal)!while!generating!another!beam!(idler)!with!
photon!energy!satisfying!energy!conservation.!

!

!ω p = !ω s + !ωi !

!

!

!

!

!

!

!

(2.1)!

!

It!must!also!fulfill!momentum!conservation!imposed!by!a!strict!phaseVmatching!
condition.!

!

!

!

!

!

!

!

!

!

!

!k p = !ks + !ki !!

!

!

!

(2.2)!

Here,!k!represents!the!wave!vectors!of!participating!beams.!!Exploiting!these!
conditions,!mixing!pump!and!signal!beams!at!a!finite!angle!can!amplify!extremely!
wide!bandwidths!(100s!of!nm)!across!the!visible!spectral!region.!!This!method!is!
known!as!Noncollinear!Optical!Parametric!Amplification!(NOPA).!!With!proper!
selection!of!crystal!angle,!crossing!angle,!and!dispersion,!output!in!the!visible!with!
bandwidths!of!100s!of!nm!corresponding!to!pulse!widths!of!a!few!femtoseconds!
with!energies!of!several!µJ!are!possible124.!
The!NOPA!used!in!this!project!was!designed!with!primary!consideration!for!
output!tunable!between!550V650!nm!with!variable!bandwidth.!!A!diagram!of!the!
beam!path!is!presented!in!Figure!2.4!and!a!brief!outline!of!its!operation!now!follows.!!
First,!a!5%!fraction!of!the!compressed!amplifier!output!is!split!off,!reflected!off!a!
controllable!delay!stage,!and!focused!into!a!sapphire!window!(2!mm!thickness)!to!

34!
!
generate!a!white!light!continuum!(signal).!!The!continuum!is!collimated,!routed!to,!
and!eventually!refocused!into!the!second!BBO!crystal.!!The!stronger!800!nm!fraction!
is!then!focused!into!a!BBO!crystal!(2!mm!thickness,!29.2°)!to!generate!the!400!nm!
pump!beam.!!This!was!then!routed!and!focused!into!BBO2.!!When!the!pump!and!
continuum!beams!have!sufficient!spatial!and!temporal!overlap,!the!NOPA!beam!
emits!in!the!proper!phase!matching!direction!along!kWL.!!The!output!is!collimated!
with!CM2!and!routed!to!the!compressor!with!flat!silver!mirrors.!
We!relied!on!a!few!parameters!to!achieve!tunable!NOPA!output.!!First,!center!
wavelength!depends!sharply!on!the!temporal!overlap!between!the!pump!and!
continuum!beams.!!Appreciable!output!typically!ranges!from!450V700!nm!in!the!
present!configuration.!!Varying!the!bandwidth!is!possible!by!tweaking!the!
dispersion!of!the!continuum!beam.!!We!introduced!deliberate!chirp!with!a!flat!
window,!W1,!composed!of!either!fused!silica!or!BK7!with!variable!thickness!(1V6!
mm).!!This!configuration!enabled!a!bandwidth!tunable!from!10!to!>100!nm!
dependent!on!selection!of!material!and!thickness.!!The!output!also!generally!
becomes!more!irregularly!shaped!with!increasing!bandwidth.!!!!

35!
!

From Regen:1 W @ 20 KHz
800 nm
50 fs

95T 5R BS

L1

M1

M5

M4

M7

M11

CM1

To NOPA Compressor

W1

M9
M6

M10

Sapphire

BBO2
BBO1

L2
Iris

M8

CM2

L3

M2

D1

M3

!

Figure(2.4.!!NOPA!beam!diagram!used!to!generate!tunable!broadband!femtosecond!
light!at!visible!wavelengths.!!!!
3.2 mm Fused Silica

6.4 mm BK7
1.2

1

1

1

0.8

0.6

0.4

0.2

Normalized Amplitude (a.u.)

1.2

Normalized Amplitude (a.u.)

Normalized Amplitude (a.u.)

No Chirp
1.2

0.8

0.6

0.4

0.2

0
540

560

580

600

620

640

Wavelength (nm)

660

680

700

720

0.6

0.4

0.2

0
520

0.8

0
520

540

560

580

600

620

640

Wavelength (nm)

660

680

700

720

520

540

560

580

600

620

640

Wavelength (nm)Figure(2.5.!!Typical!NOPA!spectral!output!with!various!white!light!chirp!as!a!
function!of!pumpVseed!delay.!!!
(
(

660

680

700

!

36!
!
2.2(Ultrafast(Pulse(Compression(
Control!of!temporal!pulse!envelope!is!an!important!consideration!for!our!
timeVresolved!experimentation.!!This!is!easily!conceived!for!a!simple!transient!
absorption!experiment,!where!experimental!time!resolution!is!limited!by!the!pulse!
duration!of!the!probe!beam.!!For!multidimensional!experimentation,!however,!these!
pulse!overlap!effects!are!manifest!in!less!predictable!ways!and!must!be!mitigated!to!
enable!proper!interpretation.!!This!section!briefly!details!the!fundamentals!of!
materialVinduced!dispersion!and!the!pulse!compression!strategies!utilized!
throughout!this!experimentation.!!!
A!wavelength!dependent!refractive!index!has!a!significant!influence!on!
broadband!pulses!necessary!for!ultrafast!spectroscopy.!!This!effect!is!commonly!
expressed!as!a!Taylor!Series!expansion!of!the!spectral!phase!as!a!function!of!
frequency.!
2

ϕ [ω − ω 0 ]ϕ (ω ) = ϕ 0 + ϕ1 [ω − ω 0 ] + 2+... !
2!

!

!

(2.3)!

The!second!order!term!is!frequency!dependent!and!represents!the!Group!
Velocity!Dispersion!(GVD)!or!its!analogue!Group!Delay!Dispersion!(GDD),!an!
intensive!quantity!encompassing!the!material!transmission!length.!!The!cubic!and!
quartic!terms!also!contribute!for!large!bandwidths!and!hinder!compression!to!very!
short!pulse!durations!as!well!as!potentially!distort!spectroscopic!results125.!
Several!strategies!exist!to!compensate!material!dispersion!in!ultrafast!
experiments.!!The!central!principle!of!each!relies!on!generating!negative!dispersion!

37!
!
equal!and!opposite!that!accumulated!through!transmissive!optical!components!
leading!to!the!sample!position.!!!
Prism#Pairs#
A!commonly!employed!method!of!dispersion!compensation!exploits!angular!
dispersion!induced!by!prisms!or!diffraction!gratings126V127.!!For!example,!a!typical!
compressor!contains!a!pair!of!Brewster!angle!cut!prisms!arranged!as!depicted!in!
Figure!2.6.!!!

Roo

P2

f Mir

ror

Lsep

P1

Lprism!
Figure(2.6.!!Prism!pair!compressor!beam!path!used!to!compress!NOPA!output.!!
The!GDD!accumulated!throughout!this!excursion!can!be!partitioned!into!
contributions!from!the!prism!separation!(Lsep)!and!transmission!through!prism!
material.!!
2d 2ϕ
λ03 # dn &λ03 d 2 n
%
(
≈
−4L
+
L
! !
sep
prism
dω 2 ω0
2π c 2 %$ d λ λ0 ('2 π c 2 d λ 2 λ0(2.4)!

The!sign!of!the!second!term!in!Equation!2.4!is!positive!in!the!visible!spectral!region.!!
Hence,!precise!insertion!of!prism!material!(Lprism)!together!with!adjusting!prism!
separation!provides!a!means!of!tuning!pulse!compression.!!The!expression!above!

38!
!
treats!only!the!quadratic!phase.!!However,!to!generate!the!shortest!possible!pulse,!
one!must!also!consider!higher!order!terms.!!Since!prisms!and!gratings!have!opposite!
relative!signs!of!cubic!phase,!a!common!strategy!is!to!employ!them!in!tandem127.!!
Although,!this!method!is!hindered!by!the!low!diffraction!efficiency!of!common!
reflection!gratings!and!tedious!alignment!required!to!achieve!optimal!compression.!!!
Chirped#Mirrors#
Recent!advances!in!thin!film!coating!technology!have!accelerated!the!
evolution!of!dispersion!compensating!mirrors128.!!Such!devices!are!beneficial!since!
they!offer!simultaneous!correction!of!GDD!and!higher!order!dispersion!with!very!
high!reflectivity.!!In!this!thesis,!a!chirped!mirror!pair!(Venteon!DCM10)!was!used,!
which!provided!V100!fs2!GDD!per!bounce!with!TOD!optimized!for!fused!silicaVtype!
glasses.!!The!spectral!dispersion!curve!for!each!mirror!is!presented!in!Figure!2.7.!!
Venteon DCM10 Measured Dispersion
200

100

GDD (fs2)

0

−100

−200

−300

−400
520

540

560

580

Wavelength (nm)

600

620

640

(

Figure(2.7.!!Dispersion!curves!for!Venteon!DCM10!chirped!mirror!pair.!!The!black!
and!grey!curves!reflect!the!measured!GDD!of!the!individual!mirrors!and!the!dotted!
blue!curve!represents!the!combined!dispersion.!!!

39!
!
To!achieve!compensation!for!all!optical!elements!before!the!sample!position,!several!
bounces!are!required!separated!vertically!in!a!doubleVpass!fashion!as!illustrated!in!
Figure!2.8.!!!

To JILAMONSTR
CM1

CM2

From NOPA(
Figure(2.8.!!Beam!path!diagram!for!doubleVpass!alignment!of!the!chirped!mirror!
pair.!!The!reverse!pass!(lighter!shade)!is!vertically!separated!with!a!roof!mirror,!
picked!off!with!a!flat!silver!mirror,!and!sent!towards!the!JILA!MONSTR!input.!!!
The!compressed!NOPA!output!was!characterized!with!Frequency!Resolved!
Optical!Gating!(FROG).!!Such!methods!allow!for!extraction!of!pulse!intensity!and!
phase129.!!This!information!not!only!verifies!proper!compression,!but!also!can!be!
used!to!account!for!experimental!artifacts!in!model!calculations.!!Transient!Grating!
FROG!(TG!FROG)!is!the!most!convenient!diagnostic!at!the!sample!position!given!the!
BOXCAR!phaseVmatching!geometry.!!However,!the!Second!Harmonic!Generation!
analogue!(SHG!FROG)!was!also!used!(Mesa!Photonics!videoFROG)!as!a!comparative!
measurement.!!The!qualitative!difference!between!these!techniques!is!that!TG!FROG!
is!χ(3)!and!can!distinguish!the!sign!of!pulse!chirp,!whereas!SHG!FROG!is!time!

40!
!
ambiguous129V130.!!Representative!TG!FROG!data!for!the!compressed!NOPA!output!is!
displayed!in!Figure!2.9.!!!!!
500
520
540

Wavelength (nm)

560
580
600
620
640
660
680
700

−150

−100

−50

0

Delay (fs)

50

100

150

!

Figure(2.9.!!TG!FROG!measured!from!the!nonresonant!signal!of!neat!benzene!
collected!at!sample!position.!!Compression!was!carried!out!with!multiple!bounces!
off!a!chirped!mirror!pair.!!!
Adaptive#Pulse#Shaping#
Adaptive!pulse!shaping!is!another!means!of!pulse!dispersion!compensation.!!
Successful!pulse!compression!has!been!demonstrated!across!a!wide!spectral!region!
with!several!different!technologies131V134.!!Moreover,!this!approach!has!been!utilized!
to!facilitate!multidimensional!spectroscopies!in!the!IR!and!visible!regimes108,!135V137.!!!
For!optical!wavelengths,!typical!strategies!involve!a!4f!configuration!where!the!
incident!light!is!dispersed!horizontally!with!gratings!or!prisms!and!dispersion!
induced!with!an!acoustoVoptic!modulator!(AOM)!or!spatialVlight!modulator!(SLM).!!
Common!pitfalls!for!each!include!limited!dynamic!range,!low!throughput,!and!

41!
!
inadequate!spectral!resolution.!!The!effect!on!spectroscopic!data!have!also!been!
addressed138.!
Adaptive!pulse!shaping!was!carried!out!with!a!linear!12288!pixel!Liquid!
Crystal!on!Silicon!(LCoS)!SLM!acquired!from!Boulder!Nonlinear!Systems.!!The!
incident!light!was!dispersed!with!a!reflective!holographic!plane!diffractive!grating!
[333!grv/mm,!Richardson!#33009FL01V321R]!as!depicted!in!Figure!2.10.!!!

333 grv/mm

l=18 mm

BNS 1D-12K LCoS SLM

From NOPA
To JILA MONSTR
Wedgesf=50 cm

Input Beam Diameter-3 mm
Throughput-65%!

Figure(2.10.!!Beam!diagram!of!the!LCoS!adaptive!pulse!shaper.!!!!!
A!beam!diagram!for!the!adaptive!pulseVshaping!device!is!presented!in!Figure!2.10.!!
In!short,!the!uncompressed!visible!pulse!train!exiting!the!NOPA!light!source!is!
aligned!into!the!diffraction!grating!at!near!the!blaze!angle!(Θ=5.7°)!to!ensure!most!
efficient!reflection.!!The!1st!order!component!is!collimated!in!the!horizontal!
dimension!with!a!concave!silver!mirror!(50!cm!focal!length,!2”!diameter),!incident!
slightly!above!its!center!axis.!!This!mirror!is!tilted!downward!slightly!to!enable!

42!
!
pickoff!with!a!flat!rectangular!silver!turning!mirror.!!The!SLM!is!positioned!for!
normal!incidence!at!the!focal!plane.!!The!SLM!contains!high!reflectance!dielectric!
coating!optimized!for!visible!wavelengths.!!Subsequent!retroreflection!follows!a!
parallel!path!offset!vertically!to!allow!for!transmission!above!the!input!mirror.!!Total!
throughput!is!roughly!65%,!comparable!or!exceeding!values!reported!for!similar!
instruments131.!!A!higher!throughput!of!>80%!can!be!achieved!by!substituting!
prisms!as!the!dispersive!element.!!!!
The!optimization!itself!is!guided!by!a!reference!signal!monitored!in#situ!and!
sensitive!to!the!pulse!duration.!!A!common!strategy!exploits!the!nonlinearity!of!
Second!Harmonic!Generation!(SHG)!on!the!shaper!output.!!A!general!limitation!of!
this!approach!is!the!finite!phaseVmatching!bandwidth!of!typical!doubling!crystals.!!
An!alternative!strategy!relies!on!the!twoVphoton!response!of!a!photodiode,!which!
provides!a!spectrally!neutral!diagnostic!for!adaptive!compression!optimization.!!
Moreover,!their!broad!spectral!response!provides!a!more!costVeffective!method!
compared!to!implementation!of!thin!BBO!crystals.!!In!this!effort,!a!silicon!carbide!
photodiode!(JECV0.1s,!EOC!Inc)!was!employed!without!the!protective!glass!cover!to!
avoid!additional!dispersion.!!This!signal!was!sent!through!a!transimpedance!
amplifier!and!sampled!with!a!NationaI!Instruments!DAQ!board!(BNCV2090A).!!This!
configuration!provided!a!stable!~DC!measurement!of!pulse!compression!
independent!of!laser!shot!noise.!!A!signal!enhancement!of!approximately!3Vfold!was!
observed!for!a!typical!compression!optimization!run.!!

43!
!
Pulse!compression!was!carried!out!with!two!separate!optimization!
algorithms.!!The!first!is!a!standard!Genetic!Algorithm!(GA)!following!random!
polynomial!generation!as!is!typical!in!other!reports.!!Convergence!with!this!method!
was!prohibitively!slow!and!unreliable!for!efficient!implementation.!!More!consistent!
results!were!realized!with!a!Freezing!Algorithm!(FA)!where!optimization!proceeds!
through!modulation!of!discrete!pixel!groups!in!a!stepwise!iterative!fashion.!!This!
method!is!outlined!in!the!flow!chart!below.!

Select Input Parameters
Pixel Groups
Phase Steps
Averages
Iterations
Interpolation

Power On and Intialize SLM

For all phase steps

For all pixel groups

Step phase of current pixel group

Apply interpolated phase mask

Read reference signal
Choose maximum

Move to next pixel group
Start in center
Alternate to outside extremes

Write optimized phase!

Figure(2.11.!!Freezing!algorithm!utilized!for!adaptive!pulse!compression.!!!
For!a!typical!FA!optimization,!best!results!were!realized!with!the!following!input!
parameters:!
Pixel!Groups! !

!

30!

Phase!Steps!per!Group!

10!

Averages!per!step!

!

3!

Number!of!Iterations!!

3!

44!
!
Interpolation! !

!

Cubic!

Starting!Phase!

!

Flat!

Spectral!resolution!is!an!important!quantity!to!consider!when!evaluating!the!
limits!of!a!given!pulse!shaping!application.!!Simultaneous!and!independent!control!
of!both!phase!and!amplitude!is!a!powerful!advantage!for!maximum!utilization!in!
spectroscopic!experimentation.!!The!latter!is!achieved!only!in!the!limit!where!a!
single!frequency!unit!is!subject!to!sufficient!pixel!coverage!enabling!application!of!
diffractive!patterns!on!top!of!the!continuous!dispersive!mask.!!This!relationship!is!
generally!described!by!the!following!relationship134!

*# 2π x &M (x) = exp ,iΔ ( x ) sin %( + iΦ ( x )/ !
$ Λ '
+
.

!

(2.5)!

Amplitude!shaping!is!possible!when!the!grating!period!(Λ)!is!small!relative!to!the!
frequency!of!the!light.!!Spectral!resolution!varied!slightly!with!bandwidth!in!this!
project.!!An!approximate!value!assuming!a!typical!light!spectrum!(600/50!nm!
FWHM)!is!0.008!nm!per!pixel!for!this!device.!!This!resolution!should!greatly!exceed!
the!limit!required!for!independent!amplitude!and!phase!control.!!!
SLM!control!was!conducted!with!various!Matlab!scripts!utilizing!library!
functions!(12kPCIe16Board.dll)!provided!by!the!manufacturer.!!Credit!is!given!to!
KapteynVMurnane!lab!members!Matt!Seaberg,!Elisabeth!Shanblatt,!Ming!Chang,!and!
Daniel!Adams!for!preliminary!development!of!this!control!software.!!Primary!
functions!act!to!initialize,!uninitialize,!and!apply!an!arbitrary!phase!mask.!!The!
following!functions!were!used!to!conduct!an!optimization!with!the!freezing!
algorithm:!!

45!
!
FreezingGUI2.m!
FreezingFcn2.m!
FreezingFitness2.m!
Representative!pulse!shaping!results!will!now!follow.!!The!input!light!used!in!
this!experiment!was!centered!at!620/55!nm!FWHM!and!precompressed!with!a!
prism!compressor!yielding!a!pulse!with!mostly!residual!TOD.!!Similar!results!were!
realized!without!precompression,!although!in!general,!convergence!consistency!
seemed!to!scale!inversely!with!starting!pulse!duration.!!Initial!optimizations!began!
with!a!flat!phase.!!Subsequent!iterations!were!seeded!with!the!most!consistent!
solution.!!However,!little!improvement!was!observed!using!this!strategy.!The!phase!
solution!for!compression!of!the!NOPA!output!is!shown!in!Figure!2.12.!!!!!!
7
6
5
Phase (Rad)

4
3
2
1
0
-1
-2
-3

0

5000
Pixel

10000

(

Figure(2.12.!!Phase!obtained!upon!optimization!with!freezing!algorithm!with!30!
pixel!groups.!!!!
The!compressed!light!was!characterized!with!TG!FROG.!!These!results!are!presented!
in!the!Figures!below.!!A!shorter!pulse!duration!is!evident!examining!the!FROG!data!
upon!optimization.!!Compensation!of!the!TOD!shoulder!was!also!clear!on!the!

46!
!
positive!time!side!in!the!TG!FROG.!!However,!there!is!interference!observed!within!
the!spectral!envelope!that!warrants!additional!discussion.!
700

a

680

680

660

660

Wavelength (nm)Wavelength (nm)

700

640
620
600
580

640
620
600
580

560

560

540

540

520
−200

−100

0
Delay (fs)

100

520
−200

200

b

−100

0
Delay (fs)

100

200

!

Figure(2.13.!!TG!FROG!before!(a)!and!after!(b)!optimization!with!freezing!algorithm.!
Before
After

1

0.8

0.6

0.4

0.2

0
−300

−200

−100

0

Delay (fs)

100

200

300

!

Figure(2.14.!!Integrated!TG!FROG!signal!before!(blue)!and!after!(red)!optimization.!
Since!third!order!methods!are!intrinsically!time!unambiguous,!the!TG!FROG!
results!demonstrate!the!compensation!of!third!order!chirp.!!In!particular,!the!
shoulder!observed!in!integrated!FROG!spectrum,!is!evidence!of!this!effect.!!There!

47!
!
still!remains,!however,!a!small!observable!shoulder!at!small!positive!delays!upon!
optimization.!!In!the!end,!the!pulse!duration,!assuming!a!Gaussian!envelope,!was!
reduced!from!26.7!to!16.8!fs.!!The!SHG!FROG!results!were!in!agreement!and!the!
extracted!spectral!phase!reveals!apparent!residual!high!order!dispersion!following!
adaptive!optimization.!!Unfortunately,!a!similar!quantification!of!spectral!phase!
from!TG!FROG!is!not!possible!within!a!homodyne!detection!scheme.!!Future!efforts!
will!concentrate!on!improving!this!measurement.!!!!!
TG!scans!were!performed!on!nile!blue!in!acetonitrile!to!explore!the!effect!of!
pulse!compression!on!an!electronically!resonant!sample!and!to!compare!with!
previous!experimental!results.!!The!data!agrees!with!previous!measurements!
compressed!with!a!prism!pair,!however,!anomalous!spectral!distortions!are!
observed!within!the!pulse!overlap!region!at!subVpicosecond!delays.!!!!!
700

a

b

Wavelength (nm)Wavelength (nm)

700

650

600

650

600

550

0

500

Delay (ps)

1000

550
0

0.2

0.4

0.6

Delay (ps)0.8

Figure(2.15.!!Nile!Blue!in!acetonitrile!TG!spectrum!following!compression!with!
freezing!algorithm!at!late!(a)!and!early!(b)!delays.!!!
This!contrast!is!even!greater!when!compared!to!data!collected!with!the!SLM!off!
(Figure!2.16)!where!obvious!spectral!distortions!are!observed.!!This!complex!

1

!

48!
!
interference!pattern!in!frequency!suggests!an!equally!distorted!temporal!profile!as!
shown!in!Figure!2.17.!!
650aWavelength (nm)Wavelength (nm)

650

600

550
0

0.05

Delay (ps)

0.1

b

600

550
0

0.15

0.05

Delay (ps)

0.1

0.15

!

Figure(2.16.!!Nile!blue!TG!data!at!early!delay!and!respective!frequency!slices!with!
SLM!off!(a)!and!on!(b).!!!
a

1
0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0
550

600

Wavelength (nm)

650

550

c

1

600

Wavelength (nm)

650

d

1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0
-1

b

1

0
-0.5

0

Time (ps)

0.5

1

-1

-0.5

0

Time (ps)

0.5

1

!

Figure(2.17.!!Frequency!and!temporal!representations!in!the!pulse!overlap!region!
of!a!nile!blue!TG!spectrum!with!SLM!off!(a,c)!and!on!(b,d).!

49!
!
Time!domain!representations!of!the!spectral!envelope!reveal!striking!differences!
whether!the!SLM!is!powered!on.!!When!powered!off,!the!primary!interference!
occurs!between!the!fundamental!and!significant!contribution!at!~35!fs!followed!by!a!
smaller!contribution!at!~68!fs.!!When!the!SLM!is!powered!on,!the!time!spectrum!
becomes!much!more!congested!with!several!minor!components!evident!out!to!
nearly!500!fs.!!!
The!enhancement!of!this!effect!for!resonant!signals!is!clear!when!applying!a!
periodic!phase.!!Figure!2.18!illustrates!the!effect!of!sinusoidal!phase!with!5Vradian!
amplitude!on!resonant!(nile!blue)!and!nonresonant!(benzene)!samples.!!

Resonant
Nonresonant

1
0.8
0.6
0.4
0.2
0
550

600

Wavelength (nm)650!

Figure(2.18.!!Spectral!envelope!following!application!of!a!sinusoidal!phase!on!
resonant!(nile!blue)!and!nonresonant!(benzene)!samples.!!!
The!results!reported!above!demonstrate!independent!phase!and!amplitude!
control!with!an!adaptive!pulse!shaper.!!No!previous!reports!of!this!application!are!
known!for!a!reflective!LCoS!SLM!at!visible!wavelengths.!!Moreover,!the!65%!
throughput!represents!a!2V3!fold!improvement!over!transmissive!liquid!crystal!

50!
!
devices.!!Unfortunately,!lingering!hardware!issues!compromised!its!full!utilization!in!
nonlinear!experimentation.!!!
The!anomalous!temporal!interference!reported!above!is!likely!related!to!
diffraction!observed!across!the!beam!following!reflection!off!the!SLM.!!A!visible!
change!is!observed!when!toggling!the!SLM!power!on/off!where!seemingly!discrete!
orders!of!stray!light!transform!into!a!near!continuum!of!light!overlapping!with!the!
fundamental!zerothVorder!beam!(Figure!2.19).!!Consultation!with!the!manufacturer!
led!a!collaborative!effort!to!identify!the!cause!of!this!issue.!!!!!!

ab!
Figure(2.19.!!Photographs!of!light!reflected!off!the!SLM!with!power!off!(a)!and!on!
(b).!!!
Upon!further!inspection,!BNS!discovered!a!similar!diffractive!effect!with!
other!devices!and!its!extent!was!seemingly!variable!and!random.!!They!also!
identified!a!periodic!32Vpixel!aberration!within!the!silicon!layer!containing!the!

51!
!
electrode!elements!that!possibly!correlated!with!its!presence.!!Unable!to!devise!an!
immediate!remedy!for!this!fabrication!dilemma,!a!couple!possible!solutions!were!
attempted.!!First,!BNS!replaced!the!original!device!with!one!demonstrating!less!of!
the!anomalous!diffraction!behavior.!!This!substitution!had!a!notable!improvement!in!
the!resonant!FWM!spectrum!shown!in!Figure!2.20.!!!

a

1
0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0
500

b

1

0
520

540

560

Wavelength (nm)

580

-0.5

0

Time (ps)0.5(

Figure(2.20.!!FWM!spectral!envelope!and!corresponding!temporal!spectrum!for!a!
second!SLM.!!
Unfortunately,!the!magnitude!of!this!improved!interference!still!exceeds!that!
necessary!for!facile!utilization!in!nonlinear!experimentation.!!A!second!proposed!
solution!involved!in#situ!electronic!compensation!of!this!periodic!aberration!by!
application!of!an!inverse!grating!pattern.!!However,!little!or!no!improvement!was!
realized!with!this!strategy.!!Finally,!a!spatial!filter!(50!µm)!was!introduced!between!
the!pulse!shaper!and!sample!position!to!help!separate!extraneous!diffracted!light!
from!the!fundamental!excitation!beam.!!Partial!improvement!was!observed,!but!not!
of!sufficient!quality!for!experimental!utilization.!!!

52!
!
1

0.8

0.6

0.4

0.2

0

550

600

Wavelength (nm)650!

Figure(2.21.!!Spectral!envelope!of!nile!blue!FWM!signal!with!spatiallyVfiltered!
excitation.!!!
We!discovered!that!temporal!pulse!structure!has!a!significant!influence!on!
the!shapes!of!peaks!our!2D!spectra.!!This!is!especially!true!at!small!delays!when!the!
pulses!partially!overlap!along!the!T!and!!"time"dimensions.""As"such,"accurate"characterization"of"pulse"phase"is"essential"for"minimizing"these"distortions"and"correct"interpretation"of"spectral"features."2.3(JILA(MONSTR(Nonlinear(Optical(Platform(
Multidimensional!spectroscopy!demands!subcycle!phase!stability!to!ensure!
accurate!detection!of!the!signal!field.!!This!becomes!even!more!critical!at!optical!
frequencies!where!ambient!mechanical!fluctuations!induce!appreciable!phase!drift.!!
Several!phase!stabilization!strategies!have!been!devised!to!mitigate!this!problem.!!
Passive!methods!rely!on!common!optical!paths,!compact!experimental!design,!and!
robust!mechanical!construction107.!!However,!these!apparatus!are!often!hindered!by!
limited!temporal!range!and!restricted!experimental!resolution.!!This!is!a!particular!

53!
!
challenge!for!biological!systems!where!the!slowest!vibrations!fall!outside!of!the!
typical!detection!limit.!!Alternatively,!active!phase!stabilization!schemes!utilize!
feedback!loops!or!pulse!shapers!to!control!pulse!phase!in#situ108,!135.!!These!methods!
are!also!inhibited!by!experimental!geometry,!restricted!temporal!range,!and!related!
considerations.!!!
Nonlinear!spectroscopic!experimentation!in!this!project!was!conducted!with!
the!JILA!Multidimensional!Optical!Nonlinear!SpecTRometer!(MONSTR)106.!!This!
instrument!was!developed!as!an!activelyVstabilized!nonlinear!optical!platform!
capable!of!generating!4!phaseVlocked!femtosecond!pulses!in!a!noncollinear!
BOXCARs!geometry.!!Moreover,!it!operates!across!a!wide!spectral!range!(400V800!
nm)!with!appropriate!optical!components!and!can!access!population!delays!of!>1!ns!
making!it!a!versatile!tool!for!conducting!multidimensional!spectroscopy.!!The!
internal!configuration!of!this!apparatus!is!outlined!in!Figure!2.22.!!!

54!
!

(
(

BottomTop(
Figure(2.22.!!CAD!drawings!of!JILA!MONSTR!assembly!and!beam!paths.!!!
!

55!
!

!
Figure(2.23.!!Photographs!of!JILA!MONSTR!nonlinear!optical!spectroscopic!platform.!
From NOPA Compressor

Error Signal Detection
Top
Bottom
Inter

M2

M3
DCM1

To Spectrometer

M1

JILA MONSTR
Gradient ND Filter

SampleReplica Focus

A2

A1
532 nm Reference!
Figure(2.24.((Beam!path!outside!of!JILA!MONSTR.!!!
Active#Phase#Stabilization##Interferometric!detection!of!the!signal!field!requires!control!of!pulse!time!
delays!with!subcycle!precision.!!At!visible!wavelengths,!this!equates!to!roughly!a!10s!

56!
!
to!100s!of!nanometer!lengthscale,!which!is!on!the!order!of!ambient!mechanical!
fluctuations!and!can!potentially!compromise!detection!and!ultimately,!spectral!
interpretation.!!Several!methods!have!been!used!to!mitigate!this!effect.!!Passive!
phase!stabilization!is!accomplished!by!compact!mechanical!design!and!use!of!
diffractive!optics107,!109.!!The!JILA!MONSTR!incorporates!an!active!stabilization!
system!utilizing!feedback!loops!that!exert!interferometric!phase!control!along!each!
beam!path!with!piezoVdriven!mirrors.!!The!phase!drift!is!evident!by!following!the!
heterodyne!signal!interference!on!the!timescale!of!experimental!data!acquisition.!!
Figure!2.25!displays!FWM!spectra!serially!collected!over!a!5!s!interval.!!Phase!
migration!is!clear!without!utilization!of!feedback!loops!where!fluctuations!on!the!
order!of!the!signal!wavelength!are!evident.!!Activation!of!the!stabilization!system!
was!found!to!produce!a!typical!phase!stability!of!>λ/200!calculated!on!the!basis!of!
the!locked!error!signal!RMS!deviation!for!each!feedback!loop.!!!!

a

593

594

595 596 597 598
Wavelength (nm)

599

600

b

593

594

595 596 597 598
Wavelength (nm)

599

600

!

Figure(2.25.!!Error!signals!(a)!with!and!(b)!without!activation!of!the!phase!
stabilization!feedback!loops.!

57!
!
Phase#Retrieval#Procedure#
Phase!instability!can!also!compromise!extraction!of!the!absolute!signal!phase!
resulting!from!slight!imprecisions!in!pulse!timing!at!the!sample!position.!!This!
ambiguity!prevents!determination!of!absorptive!lineshapes!and!may!complicate!
physical!interpretation.!!This!challenge!is!further!compounded!for!measurements!on!
biological!systems!where!the!exact!bath!coupling!is!unknown!a#priori.!!Several!
strategies!have!been!devised!for!extracting!the!absolute!signal!phase.!!The!most!
frequently!used!method,!known!as!the!projection!slice!theorem,!equates!the!
spectrallyVresolved!transient!absorption!spectrum!at!a!given!population!time!to!the!
projection!of!a!purely!absorptive!2D!spectrum!along!the!emission!frequency!
dimension139.!!This!method!is!known!to!work!reliably!for!strongly!absorbing!
samples,!but!is!hindered!for!cases!of!low!excitation!density,!crossVpolarized!
excitation,!and!double!quantum!coherence!spectra.!!Moreover,!replicating!the!exact!
excitation!conditions!in!a!second!measurement!is!a!tedious!experimental!burden.!!
Alternative!techniques!have!been!proposed!to!overcome!these!obstacles!including!
allVoptical!procedures!that!rely!on!in#situ#correction!of!phase!drift!using!a!pulse!
shaper!or!duplication!of!the!beam!overlap!at!the!sample!position!for!more!efficient!
extraction!of!global!phase!offsets140.!!In!this!project,!a!combination!of!the!projection!
slice!technique!and!the!allVoptical!method!proposed!by!Bristow!et#al.#are!used!to!
retrieve!the!absolute!signal!phase141.!!The!SRTA!spectrum!used!for!the!former!
method!was!collected!as!described!above!before!each!respective!2D!scan.!!

58!
!

a

b

c

!
Figure(2.26.((Field!correlations!for!(a)!XY,!(b)!YU,!and!(c)!XYUZ!beam!combinations!
collected!at!the!replica!focus!position!used!in!absolute!phase!retrieval.!
Phase#Cycling#
Excitation!beam!scatter!is!a!universal!challenge!for!most!laser!spectroscopies.!!
This!is!especially!true!for!liquid!biological!samples!that!are!prone!to!aggregation!and!
cases!where!a!spinning!sample!cell!is!used!to!facilitate!data!collection.!!Heterodyne!
detection!coupled!with!interferometric!temporal!control!allows!for!implementation!
of!a!straightforward!phase!cycling!procedure!to!minimize!background!scatter!and!
improve!data!quality.!!!!
X Stage Phase Cycling
x

2

x

Nonrephasing

Rephasing
0

1

0.8

Stage Motion (µm)

Stage Motion (µm)

2

0.6

4

6

8

0.4
10

0.2

12

14

0

0

5

10

15

Step Number

20

25

16

0

5

10

15

20

25

Step Number

Figure(2.27.!!Stepping!sequence!for!XVaxis!phase!cycling!in!nonrephasing!and!
rephasing!data!collection!scans.!

!

59!
!
In!principle,!it!is!possible!to!phase!cycle!and!eliminate!scatter!contributions!
from!all!excitation!beams,!however,!it!becomes!increasingly!time!consuming!to!
account!for!all!phaseVshifted!permutations!with!each!additional!axis.!!For!this!study,!
phase!cycling!of!one!or!two!axes!was!sufficient!to!facilitate!efficient!collection!of!
high!quality!2D!data.!!The!stepping!pattern!for!this!procedure!is!described!in!Figure!
2.28.!!These!plots!illustrate!rephasing!and!nonrephasing!stepping!patterns!for!the!X!
axis.!!For!the!rephasing!response,!X!is!phase!cycled!concurrent!with!scanning!the!!"delay"(d!=2π),"while"for"the!nonrephasing!response,!X!is!idle!and!its!only!
movement!reflects!the!πCshifts"associated"with"phase"cycling.""Interferograms"are"collected"for"the"“zeroC“"and"πCshifted"cases"and"combined"to"yield"the"final"scatterC
free"spectrogram.""This"effect"is"demonstrated"in"Figure"2.28"where"the"phaseC
cycled"data"quality"is"noticeably"improved."""

4

4

x 10

−1.6

a

x 10

b

−1.65

exc

(cm−1)−1.65ΩΩexc (cm−1)

−1.6

−1.7

−1.75
1.6

1.65
1.7
Ωemis (cm−1)

1.75
4
x 10

−1.7

−1.75
1.6

1.65
1.7
Ωemis (cm−1)

1.75
4
x 10

Figure(2.28.!!Absolute!value!rephasing!2DFTS!spectrum!of!MgVmyoglobin!(a)!
without!and!(b)!with!phaseVcycled!data!collection.!!!
#
#

60!
!
Output#and#Sample#Position#
Upon!exiting!the!JILA!MONSTR,!the!excitation!beams!are!directed!to!the!
sample!position!as!illustrated!in!Figure!2.24.!!The!beams!are!first!turned!90!degrees!
with!a!flat!2Vinch!square!silver!mirror!(M1)!towards!a!2Vinch!diameter!spherical!
mirror!(focal!length=20!cm).!!A!surface!reflection!off!a!1Vinch!diameter,!1!mm!thick!
fused!silica!window!with!backside!antireflection!coating!is!back!reflected,!turned!90!
degrees,!and!focused!into!a!40x!microscope!objective,!and!detected!with!a!CMOS!
camera!(Mightex).!!This!overlap!represents!a!replica!of!the!sample!focal!plane!and!is!
used!for!beam!alignment!and!extraction!of!the!global!signal!phase!as!described!
above.!!The!converging!transmitted!beams!are!picked!off!with!a!1Vinch!diameter!
silver!mirror!and!turned!90!degrees!towards!the!sample!position.!!The!beams!are!
collimated!with!an!identical!20!cm!focal!length!2Vinch!diameter!spherical!mirror.!!
The!signal!is!picked!off!with!a!1Vinch!square!silver!mirror!and!directed!towards!the!
spectrometer!for!detection.!!!
2.4(Data(Acquisition(Procedures(
This!section!will!detail!typical!data!acquisition!procedures!for!the!various!
nonlinear!spectroscopic!experiments!utilized!in!this!thesis.!
Transient#Grating#FROG!
Transient!Grating!FROG!experiments!were!performed!by!scanning!k3!
through!a!specified!interval!about!k1(and!k2!overlapped!at!time!zero.!!The!FWM!
signal!was!homodyne!detected!with!a!liquid!N2Vcooled!CCD!camera.!!Typical!
experimental!parameters:!

61!
!
Scan!length! !

!

200!fs! !

Step!size!

!

1.7!fs!!

Integration!Time!

!

20!ms!

Averaging!

!

1!

!

!

Total!scan!duration! !

!

~1.5!minutes!

Spectrally;Resolved#Transient#Grating#Spectroscopy#
SpectrallyVResolved!Transient!Grating!(SRTG)!spectroscopy!was!performed!
by!scanning!k3!at!a!specified!positive!time!delay!with!k1(and!k2!overlapped!at!time!
zero.!!The!FWM!signal!was!homodyne!detected!with!a!liquid!N2Vcooled!CCD!camera.!!
Prior!to!the!scan,!a!background!at!V500!fs!delay!was!collected!and!subtracted!from!
the!final!data!to!compensate!for!residual!steady!state!fluorescence!and!excitation!
beam!scatter.!!Heterodyne!detection!was!used!for!measurement!of!weak!signal!
intensity.!!!
Scan!length! !

!

1.3!ns! !

Step!size!

!

varied!

Number!of!steps!

!

varied!!

Integration!time!

!

20!ms!

Averaging!

!

1!

!

!

Total!scan!duration! !

!

!

~5!minutes!

Spectrally;Resolved#Transient#Absorption#Spectrsocopy#
Transient!Absorption!(SRTA)!spectroscopy!was!performed!in!the!standard!
pumpVprobe!geometry.!!k3!and!the!local!oscillator!were!used!as!the!pump!and!probe!
beams!respectively.!!The!probe!was!attenuated!with!a!neutral!density!filter!to!an!
intensity!ratio!of!approximately!10:1.!!Pump!modulation!was!carried!out!in!one!of!

62!
!
two!possible!ways.!!In!one!method,!the!pump!beam!was!modulated!at!a!harmonic!of!
the!laser!repetition!rate!with!a!chopper!wheel!(New!Focus!3502).!!Alternatively,!this!
beam!was!blocked!with!a!computerVcontrolled!mechanical!shutter.!!Integrated!
measurements!were!detected!with!a!lockVin!amplifier!(Stanford!Research!830)!and!
photodiode!(Thorlabs!DET36A).!!SpectrallyVresolved!measurements!were!detected!
either!with!a!liquid!N2Vcooled!CCD!camera!or!a!fiberVcoupled!USB!spectrometer!
(Ocean!Optics!USB3000+).!!This!method!combined!with!synchronization!of!an!
optical!chopper!provides!the!most!sensitive!means!of!TA!experimentation.!!!!!!
3PEPS#
Stimulated!Photon!Echo!Peak!shift!data!was!collected!in!the!BOXCARs!
excitation!geometry.!!Rephasing!and!nonrephasing!responses!were!collected!in!a!
continuous!scan!by!first!starting!k2!at!positive!time!and!collecting!data!backwards!
towards!time!zero!(nonrephasing).!!The!rephasing!half!followed!by!collecting!data!
forward!in!time!stepping!k1.!!This!strategy!helps!mitigate!time!missteps!associated!
with!acquiring!the!complete!response!in!two!separate!(rephasing!and!
nonrephasing)!halves.!!SpectrallyVresolved!heterodyne!or!homodyne!detection!was!
carried!out!with!a!liquid!N2Vcooled!CCD!camera!dependent!on!the!signal!strength.!!
Phase!stabilization!was!utilized!for!the!case!of!heterodyne!detection.!!The!integrated!
FWM!signal!versus!τ!was!fit!to!a!suitable!function.!!The!peak!shift!was!determined!
by!extracting!its!center!position.!!Previous!reported!electronic!3PEPS!measurements!
employed!a!triangular!excitation!beam!geometry!where!the!rephasing!and!
nonrephasing!signals!emit!in!unique!phaseVmatching!directions101,!142V144.!!Whereas,!
vibrational!echo!experiments!carried!out!in!the!BOXCARs!geometry!have!been!

63!
!
previously!reported102,!145.!!To!measure!the!solvation!correlation!function!and!
relative!inhomogeneity,!the!peak!shift!was!collected!as!a!function!of!population!time!
(T).!!Since!our!population!time!interval!is!limited!to!~1.3!ns,!all!dynamics!occurring!
beyond!this!were!considered!static.!!!
Scan!length! !

!

varied!!

Step!size!

!

2!fringes!

Number!of!steps!

!

varied!!

Integration!time!

!

10V20!ms!

Averaging!

!

1!

!

!

Total!scan!duration! !

!

!

~2!minutes!

Multidimensional#Spectroscopy#
TwoVdimensional!single!and!double!quantum!coherence!data!was!collected!
in!the!BOXCARs!excitation!geometry.!!Rephasing!and!nonrephasing!responses!were!
collected,!as!with!3PEPS,!in!a!continuous!scan!by!first!starting!k2!at!positive!time!
and!collecting!data!backwards!towards!time!zero!(nonrephasing).!!The!rephasing!
half!followed!by!collecting!data!forward!in!time!stepping!k1.!!Spectrally!resolved!
heterodyne!detection!was!employed!with!a!liquid!N2Vcooled!CCD!camera.!!An!
ILO:IFWM!ratio!of!4:1!was!achieved!with!neutral!density!filters!along!the!LO!beam!path!
before!combination!with!the!FWM!signal.!!Phase!stabilization!was!utilized!to!ensure!
proper!resolution!of!the!signal!field.!!PhaseVcycling!was!carried!out!to!facilitate!
direct!quantification!EFWM!without!subtraction!of!noncontributing!elements!from!the!
local!oscillator,!excitation!beam!scatter,!and!steady!state!fluorescence.!!
Quantification!of!Ωemis!was!accomplished!through!spectral!resolution!of!the!FWM!

64!
!
signal!at!each!τ!delay.!!While!Ωexc!information!was!accessed!via!subsequent!Fourier!
transformation!following!acquisition!of!the!entire!spectrogram.!!Resolution!along!
the!emission!axis!is!limited!by!the!spectrometer!and!was!~8!cmV1!at!600!nm!using!a!
300!grv/mm!dispersion!grating.!!Resolution!of!the!excitation!frequency!was!dictated!
by!step!size!and!scan!length!along!τ!in!accordance!with!the!Fourier!theorem.!!
Scan!length! !

!

varied!!

Step!size!

!

2!fringes!

Number!of!steps!

!

varied!!

Integration!time!

!

10!ms!

Averaging!

!

1!

!

!

Total!scan!duration! !

!

!

~2!minutes!

!
2.5(Sample(Preparation(Protocol(
This!section!will!describe!preparation!of!samples!utilized!throughout!this!
thesis.!!#Fluorescent#Protein#Expression#and#Purification#
Fluorescent!protein!growth!and!expression!was!conducted!in!accordance!
with!reported!accounts,!but!with!some!variation!for!select!species11,!44,!50.!!Namely,!
small!differences!in!buffer!composition!and!pH,!E.#coli!cell!strain,!and!expression!
conditions!were!tweaked!to!optimize!protein!production.!!The!following!protocol!
was!typical!for!most!RFP!derivatives.!!In!short,!a!protein!gene!was!ligated!into!the!
pBad!expression!vector!and!subsequently!transformed!into!Top10!competent!cells.!!
Transformed!competent!cells!were!cultured!on!agar!plates!(LB+ampicillin)!and!a!
starter!culture!(5V15!mL!LB!+!ampicillin)!was!grown!overnight!at!37!°C!from!a!single!
colony!pick.!!This!starter!culture!was!then!used!to!inoculate!an!expression!

65!
!
culture=50V100!mL!of!2XYT!media!(100x!dilution)!in!a!250!mL!Erlenmeyer!flask.!!
The!expression!culture!was!grown!at!37!°C!until!cloudy!(OD600~0.6)!and!
subsequently!induced!with!0.2%!arabinose!(pBad).!!The!temperature!was!then!
reduced!to!30!°C!and!expression!proceeded!for!24V48!hours.!!The!cultures!were!
harvested!by!spinning!in!a!centrifuge!at!7000!RPM!for!12!minutes!and!discarding!
the!remaining!supernatant.!!The!residual!pellets!were!preserved!at!V80!°C!for!future!
extraction!and!purification.!!Examples!of!poor!and!rich!protein!cultures!are!shown!
in!Figure!2.29.!

!
Figure(2.29.!!Photographs!of!a!(a)!low!and!(b)!high!yield!fluorescent!protein!cell!
pellet.!!!
The!cell!pellets!were!lysed!with!a!commercial!protein!extraction!agent!(BVPER,!
Thermo!Scientific)!and!centrifuged!to!remove!macroscopic!cellular!debris.!!The!
crude!product!was!then!loaded!into!a!NiVATA!column!and!incubated!for!10V15!

66!
!
minutes!in!an!ice!bath.!!Elution!of!the!purified!protein!was!carried!out!by!flowing!
buffer!containing!increasing!concentrations!of!imidazole!(10V150!mM)!in!
approximately!5!separate!rinses.!!The!final!product!was!either!refrigerated!for!
immediate!use!or!flash!frozen!in!liquidVN2!to!preserve!for!future!experimentation.!
Site#Directed#Mutagenesis#
Point!mutations!of!a!native!protein!sequence!were!used!to!systematically!
alter!specific!chromophoreVsidechain!interactions.!!QuikChange!(Agilent)!is!a!
commercially!available!kit!to!carry!out!siteVdirected!mutagenesis.!!Its!protocol!is!
outlined!in!the!Figure!below.!!!!!

Prepare native plasmid DNA

1

Design primer with desired mutant codon andcomplementary to native strand

2

Thermocycle to denature native plasmids andanneal mutant primers

3

5

Transform mutant plasmids into
E coli competent cells

6mutant codon

Perform PCR to amplify mutant plasmids

4

Digest native fragments with Dpn1 yielding nickedmutant plasmids(
Figure(2.30.!!QuikChange!siteVdirected!mutagenesis!protocol.!!!!
Following!preparation!of!mutant!protein!plasmid!DNA,!expression!and!purification!
were!carried!out!according!to!the!previously!described!procedures.!
#
#
#

67!
!
Organic#Dyes#in#Bulk#Solvent#
All!organic!dyes!and!solvents!were!used!as!received!from!the!manufacturer!
unless!noted!in!their!respective!section.!!Spectroscopic!grade!solvents!were!used!
whenever!possible!to!avoid!sample!contamination!and!experimental!interference.!!!
2.6(Auxiliary(Experimental(Details(
Sample#Cell#
The!liquid!sample!was!contained!between!two!1!inch!round!flat!windows!
separated!by!an!annular!Teflon!spacer.!!The!front!window!is!1!mm!thick!uv!fused!
silica!and!the!back!window!is!3.2!mm!thick!BK7!glass.!!The!experimental!pathlength,!
d,!was!controlled!by!the!thickness!of!the!spacer!and!ranged!from!10s!of!microns!to!1!
mm.!!This!cell!was!contained!within!an!aluminum!optic!mounting!piece!(Newport!
LPLHV1T),!which!is!attachable!to!either!a!3Vaxis!translational!mount!or!the!spinning!
sample!cell!described!below.!!!
d

3.2 mm 1 mm!

Figure(2.31.!!Cell!used!to!contain!liquid!samples!for!spectroscopic!experimentation.!!!
#
#
#

68!
!
Spinning#Sample#Cell!
Both!biological!and!dye!samples!were!spun!with!a!mechanical!spinning!cell!
for!most!data!collection.!!Refreshing!the!sample!is!necessary!to!minimize!
photobleaching,!diffusive!fluctuations!observed!in!the!FPs,!and!accumulation!of!
laserVinduced!byproducts!with!lifetimes!comparable!with!the!laser!repetition!rate.!!
These!considerations!were!evaluated!by!calculating!the!fraction!of!molecules!in!the!
excited!state!for!typical!experimental!conditions!(100!µM!concentration,!50!µm!spot!
size,!1!nJ!pulse!energy,!20!kHz!laser!repetition!rate).!!Assuming!a!cell!rotating!at!a!
rate!of!2000!revolutions!per!minute,!or!a!rotational!velocity!of!104.67!cm/s,!~1!x!10V
6!pulses!interact!with!a!given!illuminated!spot!on!average.!!This!equates!to!an!

approximate!excited!fraction!of!<0.0001%!per!spot!while!spinning,!which!should!be!
adequately!low!to!prevent!repeated!exposure!of!a!single!excited!volume.!!Another!
factor!considered!when!establishing!excitation!conditions!is!the!repopulation!rate!of!
the!electronic!ground!state.!!The!singlet!excited!state!lifetime!of!a!fluorescent!
protein!sample!falls!between!1V3!ns.!!However,!a!reversible!dark!state!conversion!
process!is!known!to!occur!in!RFPs!on!~ms!timescale146.!!Too!high!a!laser!repetition!
rate!would!lead!to!accumulation!of!photoinduced!byproducts!and!eventual!loss!of!
signal!intensity!as!is!observed!in!some!porphyrin!species!that!undergo!intersystem!
crossing.!!No!evidence!of!dark!state!conversion!was!observed!with!a!laser!repetition!
rate!of!20!kHz.!
The!photographs!below!illustrate!the!spinning!sample!cell!construction.!!The!
sample!is!contained!within!removable!piece!(Newport!LPLHV1T)!that!threads!
directly!to!the!rotating!arm.!!Rotational!velocity!was!measured!by!attaching!a!small!

69!
!
piece!of!aluminum!foil!on!the!edge!of!the!sample!cell!and!capturing!the!stray!light!
following!exposure!with!the!532!nm!reference!laser.!!Endurance!testing!revealed!a!
constant!velocity!was!maintained!for!>1!hour,!which!is!suitable!for!typical!
spectroscopic!experimentation.!!However,!there!is!a!short!stabilization!period!of!
typically!10!minutes!upon!initial!daily!usage.!!This!consistency!was!realized!
following!conversion!to!a!brushless!DC!motor!and!greatly!exceeded!that!of!the!
original,!which!experienced!intermittent!periods!of!acceleration!and!deceleration!
leading!to!baseline!drift,!which!compromised!data!collection!efforts.!
a

b

c

(
Figure(2.32.!!Photographs!of!spinning!sample!cell!from!(a)!front,!(b)!back,!and!(c)!
top!perspectives.!
Phase!stability!was!slightly!hindered!by!mechanical!vibrations!accompanying!
rotation!of!the!spinning!cell.!!This!is!evident!by!monitoring!the!FWM!signal!phase!
over!time!displayed!in!Figure!2.33.!!

70!
!

a

593

594

595 596 597 598
Wavelength (nm)

599

600

b

593

594

595 596 597 598
Wavelength (nm)

599

600

(

Figure(2.33.!!FWM!signal!fringe!phase!stability!with!spinning!cell!(a)!off!and!(b)!on.!
Spinning#cell#components#
Bearings:!

!!!!!!!!!!!!! B544DDFS428

Allied!!

Motor:!!

!

A Main Hobbies!!

EFLM4032A

E-flight 32 Brushless DC!

Power!Supply:!

10 V/1.5 Amp DC

JILA Electronic Shop!

Controller:!!! !

SPM5510A Main Hobbies

Spektrum DX5e!

Receiver:!!!

!

CSE010-0051-00

A Main Hobbies

Castle Creations Thunderbird 36-Amp Brushless ESC!

Pulleys:!

!

A6A51-072NF0608

Stock Drive Products/Sterling Instruments

Aluminum, 2 mm pitch, 0.25" bore size, 1.805" pitch diameter, 0.236" belt
width

!

!

!

A6A51-060DF0608

Stock Drive Products/Sterling Instruments

Aluminum, 2 mm pitch, 0.25" bore size, 1.505" pitch diameter, 0.236" belt
width

Belt:! !

!

A6R51M150060

Stock Drive Products/Sterling Instruments

Timing belt, neoprene, 2 mm pitch, 300 mm pitch length

!
!

71!
!
Chapter(III(Hydrogen(Bond(Flexibility(Correlates(with(Stokes(Shift(in(mPlum(Variants(
As!Appeared!in!the!Journal!of!Physical!Chemistry!B,!2014,!Vol.!118,!2940V2048!!

!
3.1(Introduction(
Fluorescent!proteins!(FPs)!derived!from!marine!organisms,!including!the!
green!fluorescent!protein!extracted!from!the!jellyfish!Aequorea#victoria,!have!
revolutionized!cellular!imaging!experiments!and!become!an!invaluable!tool!in!
molecular!biology!research147,1.!!Their!utility!is!owed!to!several!favorable!intrinsic!
properties,!namely,!tunable!excitation!and!emission,!genetic!encodability,!and!
targetability.!!Next!generation!farVred!emitting!species!with!enhanced!fluorescence!
quantum!yields!and!high!photostability!are!eagerly!sought.!!These!improvements!
would!enable!deeper!tissue!penetration!and!lengthened!imaging!times!given!a!lower!
autofluorescence!background,!lower!light!scattering,!and!higher!transmission!
beyond!650!nm14.!!!
The!fluorescent!chromophore!of!FPs!is!autocatalytically!formed!in!the!
presence!of!molecular!oxygen,!and!is!contained!within!an!elevenVstranded!βVbarrel.!!
Electronic!excitation!in!the!chromophore!occurs!with!electron!density!transferred!
from!the!phenoxy!ring!towards!the!acylimine!across!the!πVconjugated!system148V149.!!
Several!strategies!have!been!used!to!engineer!further!redVemitting!species14,!18.!!One!
approach!is!to!extend!conjugation!within!the!chromophore!itself!or!with!nearby!
sidechains!via#selective!mutagenesis.!!Alternatively,!one!can!modify!the!surrounding!
chromophore!environment!leading!to!direct!stabilization!(destabilization)!of!the!

72!
!
excited!(ground)!state!by!electrostatic!effects,!hydrogenVbonding!interactions,!or!
isomerization.!!Examples!of!these!interactions!are!found!in!several!red!FP!species,!
though!in!most!cases,!interpretation!is!derived!from!crystal!structures!and!neglects!
a!possible!dynamic!element11,!18,!47,!49.!!
ON
2.56 Å

N

O

N

E16

O

I65
!

Figure(3.1.!!Chemical!structure!of!mature!mPlum!chromophore.!!The!hydrogen!
bond!thought!responsible!for!its!large!Stokes!shift!is!shown!as!a!dashed!line!
between!E16!and!the!chromophore!terminus!of!residue!I65.!!!
The!fluorescent!protein!mPlum,!developed!through!iterative!somatic!
hypermutation!of!mRFP!(from!the!progenitor!DsRed),!has!a!peak!excitation!of!589!
nm!and!a!Stokes!shift!of!59!nm44.!!A!hydrogen!bond!between!the!E16!sidechain!and!
NVacylimine!oxygen!of!the!I65!residue!at!the!base!of!the!chromophore!as!depicted!in!
Figure!3.1!is!thought!to!be!the!key!feature!leading!to!its!large!Stokes!shift.!!This!
hypothesis!evolved!from!a!few!striking!observations:!!first,!as!illustrated!in!the!
original!report!by!Wang!et#al.,!steady!state!emission!values!vary!widely!with!point!
mutations!at!these!positions!and!all!reported!variants!show!a!nearly!20!nm!blue!
shift!relative!to!the!native!E16!configuration.!!The!Stokes!shifts!fall!within!3!distinct!
groups!around!25!nm,!35!nm,!and!50!nm,!which!may!imply!common!dynamic!
behavior!within!mutant!subgroups!of!similar!hypsochromic!shift.!!Moreover,!these!

73!
!
perturbations!occur!primarily!on!the!excited!state!given!the!sensitivity!of!the!
emission!wavelength.!!Second,!Abbyad!et#al.!identified!a!large!dynamic!Stokes!shift!
with!timescales!of!4!and!71!ps!in!mPlum!using!fluorescence!upconversion!
spectroscopy45.!!These!dynamics!differed!from!mRaspberry!and!mRFP,!which!
showed!very!little!solvation!over!the!1!ns!probe!window.!!They!explained!this!shift!
in!terms!of!a!specific!interaction!between!E16!and!I65!on!the!basis!of!their!influence!
on!the!steady!state!Stokes!shift.!!This!was!the!first!report!of!a!time!dependent!
interaction!leading!to!an!extended!red!shift!in!FPs.!!These!authors!proposed!that!the!
solvation!process!resulted!from!an!excited!state!rearrangement!of!a!specific!
interaction!between!residues!16!and!65,!yet!their!evidence!did!not!discount!the!
possibility!that!general!flexibility!of!sideVchains!or!water!around!the!chromophore!
may!be!responsible.!!More!recently,!crystal!structures!of!mPlum!together!with!E16Q!
and!I65L!variants!reaffirmed!the!hydrogen!bond!between!E16!and!I65!in!mPlum!
and!identified!a!similar!interaction!in!the!E16Q!mutant46.!!They!also!implicated!the!
steric!influence!of!I65!and!predict!it!plays!a!role!in!strengthening!this!hydrogen!
bond!in!the!excited!state.!!!!!!
Several!different!femtosecond!spectroscopy!techniques!are!capable!of!
resolving!the!time!dependent!redVshift!due!to!the!excited!state!solvation!
dynamics150,151.!!Here,!spectrally!resolved!transient!grating!spectroscopy!(SRTG)!is!
employed!as!previously!applied!by!Joo!et#al.#to!observe!nuclear!wavepacket!
dynamics!in!ground!and!excited!electronic!states!and!solvation!of!dyes!in!bulk!
solvent91,!152.!!In!this!fourVwave!mixing!technique,!two!pump!pulses!(with!
wavevectors!k1!and!k2)!interfere!imprinting!a!spatial!grating!on!the!sample!which!is!

74!
!
later!probed!by!a!third!pulse!(with!wavevector!k3)!at!a!controllable!delay!that!
diffracts!into!a!spatially!isolated!phase!matching!direction!(k1Vk2+k3).!!For!a!
chromophore!with!a!large!Stokes!shift!together!with!a!sufficient!excitation!window,!
this!signal!can!be!spectrally!resolved!into!stimulated!emission!(SE)!and!groundVstate!
bleach!(GSB)!contributions.!!Time!evolution!of!the!GSB!and!SE!bands!represent!
ground!and!excited!state!solvation!dynamics!respectively.!!
In!this!chapter,!SRTG!spectroscopy!and!molecular!dynamics!(MD)!
simulations!are!employed!to!explore!the!relationship!between!flexibility!of!the!
chromophore!environment!and!the!large!Stokes!shift!in!mPlum.!!Relaxation!of!a!
panel!of!strategic!point!mutants!of!the!participating!16!and!65!residues!was!
investigated.!!The!experimental!results!and!simulations!indicate!the!redVshifted!
emission!in!mPlum!is!correlated!with!a!facile!switching!between!a!direct!and!waterV
mediated!hydrogen!bond!between!the!E16!sidechain!and!the!NVacylimine!oxygen!at!
position!65.!!All!discussions!about!the!role!of!a!hydrogen!bond!network!in!earlier!
papers!were!derived!from!a!static!crystal!structure!in!the!ground!state.!!This!chapter!
represents!advancement!in!that!it!explores!hydrogen!bond!dynamics!around!the!
chromophore!rather!than!the!hydrogen!bond!network!derived!from!an!average!
ground!state!structure.!
3.2(Experimental(Methods(
Time;resolved#Measurements#
Transient!grating!measurements!were!performed!in!the!traditional!BOXCARs!
geometry!as!established!in!the!previously!described!JILA!MONSTR!nonlinear!optical!
platform106.!!Upon!irradiation!with!3!excitation!pulses!(20!fs,!~10!nJ/beam),!

75!
!
generated!from!a!noncollinear!optical!parametric!amplifier!pumped!with!a!
Ti:Sapphire!regenerative!amplifier!(20!kHz),!the!spatially!isolated!fourVwave!mixing!
signal!emitted!in!the!prescribed!ks=Vk1+k2+k3!phase!matching!direction.!!Excitation!
wavelength!was!chosen!to!maximize!contrast!between!ground!state!bleach!and!
stimulated!emission!components.!!!!!
Table(3.1.((Excitation!center!position!and!FWHM!bandwidth!used!in!SRTG!
measurements.!
Mutant&Wavelength&(nm)&mPlum&

625440&E16Q&

621450&E16H&

610440&E16L&615445&

I65L&

625450&I65A&

625450&I65V&

625450&!
The!sample!was!refreshed!with!a!spinning!cell!to!mitigate!photobleaching!
and!accumulation!of!laserVinduced!photoproducts.!!Sample!concentration!and!path!
length!were!carefully!tuned!to!avoid!aggregation!and!reabsorption!of!the!nonlinear!
signal.!!Concentrations!of!~50!µM!and!a!path!length!of!0.5!mm!were!used!for!all!
samples!(OD!0.15!peak!absorption).!!Spectrally!resolved!detection!was!carried!out!
with!a!liquid!N2Vcooled!CCD!camera.!!Data!was!collected!as!a!function!of!the!T!time!
delay!(k1(and!k2(temporally!overlapped)!from!a!range!of!0!to!1.3!ns,!spaced!
logarithmically,!with!a!background!spectrum!collected!at!V500!fs!delay!to!eliminate!
contributions!from!excitation!scatter!and!spontaneous!emission.!!
#
#

76!
!
Protein#Expression#and#Purification#
Fluorescent!proteins!were!expressed!with!a!pBAD!expression!vector!
containing!the!mPlum!gene.!!Point!mutations!were!accomplished!with!QuikChange!
mutagenesis!from!commercial!primers.!!Upon!sequencing!confirmation,!plasmids!
were!transformed!into!Top10!E.!coli!followed!by!induction!with!0.02%!arabinose!for!
24!hours!at!30!°C.!!Samples!were!purified!via!6XVHis!tag/NiVNTA!chromatography!
and!the!crude!specimens!were!bufferVexchanged!with!15!mM!pH!8.0!TRIS!buffer,!
100!mM!KCl.!!Emission!spectra!of!each!mutant,!excited!at!532!nm,!were!detected!
with!a!diode!array!spectrometer.!!
Molecular#Dynamics#Simulations#
Time!series!trajectories!were!obtained!from!explicit!solvent,!allVatom!
simulations!using!the!NAMD!molecular!dynamics!package!with!the!CHARMM27!
force!field153V154.!!The!initial!XVray!crystallographic!structures!were!obtained!from!
the!Protein!Data!Bank!(mPlum!pdb!code!2QLG,!E16Q!pdb!code!2QLI,!I65L!pdb!code!
2QLH).!!The!other!four!mutants!do!not!have!PDB!structures,!so!we!used!CHARMM!to!
make!the!amino!acid!substitution!in!the!mPlum!pdb!2QLG!file.!!Force!field!
parameters!for!the!mature!chromophore!were!adopted!from!the!anionic!GFP!
chromophore!developed!by!Reuter!et#al.!and!from!CHARMM27!parameters!for!
acylimine!nitrogen155.!!Throughout!the!simulation,!the!deprotonated!anionic!form!of!
the!chromophore!in!the!ground!state!was!used.!!In!addition,!E215!was!protonated!
using!a!patch.!
The!VMD!package!was!used!to!setup!the!system!for!simulations156.!!The!
initial!structure!of!mPlum!with!crystallographic!water!molecules!was!solvated!by!

77!
!
using!the!solvate!plugin!in!VMD.!!For!all!mutants,!the!box!cutoff!was!set!to!10!Å.!!For!
mPlum,!this!resulted!in!a!simulation!box!of!dimensions!65.1!x!62.6!x!71.7!Å3,!with!
similar!dimensions!for!the!other!mutants.!!The!solvated!system!was!electrically!
neutralized!by!adding!five!Na+!ions!randomly!in!the!bulk!water!using!the!VMD!
autoionize!plugin.!!The!final!system!contained!a!total!of!27188!atoms.!!All!water!
molecules!overlapping!with!the!protein!were!removed.!!The!particle!mesh!Ewald!
method!was!used!to!treat!longVrange!interactions!with!a!12!Å!nonbonded!cutoff157.!!
Energy!minimization!was!performed!using!the!conjugate!gradient!and!line!search!
algorithm.!!The!system!was!then!heated!for!90!ps!with!a!linear!gradient!of!20K/6ps!
from!20!to!300!K.!!At!300!K,!the!system!was!equilibrated!for!910!ps!with!a!2!fs!
integration!time!step!in!the!NVT!(constant!number,!volume,!and!temperature)!
ensemble.!Langevin!dynamics!was!used!to!maintain!the!temperature!at!300!K.!!The!
production!run!was!50!ns!using!NVT!dynamics!with!2!fs!time!steps.!!The!last!40!ns!
of!the!production!run!was!used!for!analysis.(
3.3(Results(
Steady#State#Spectroscopy#
Linear!absorption!and!emission!peaks!for!each!mutant!are!shown!in!Table!
3.2.!!The!absorption!peaks!fall!within!a!narrow!range!around!590!nm!aside!from!
E16L,!E16H,!and!I65L,!which!exhibit!~5!nm!blue!and!red!shifts!respectively.!!The!
Stokes!shifts!range!from!1600!cmV1!and!778!cmV1!with!mPlum!and!E16L!being!the!
largest!and!smallest!respectively.!!
(
(

78!
!
Table(3.2.(!Steady!state!absorption!and!emission!data!for!each!mutant.!
Mutant&Absorption&Peak&(nm)&

Emission&Peak&(nm)&

Stokes&Shift&41(cm )&

mPlum&

589&

648&

1593&

E16Q&

588&

630&

1134&

E16H&

586&

620&

936&

E16L&

586&

614&

778&

I65L&

596&

629&

881&

I65A&

591&

630&

1047&

I65V&

591&

641&

1319&

!
Time;resolved#Measurements#
To!differentiate!the!solvation!dynamics!of!the!variants,!the!spectral!shape!
and!time!evolution!of!the!transient!grating!signals!was!analyzed.!!SRTG!spectra!of!
mPlum!and!the!I65L!for!various!time!slices!are!displayed!in!Figure!3.2a.!!The!results!
for!these!mutants!were!plotted!to!illustrate!the!range!of!behavior!among!all!species.!!!

79!
!

1

a

3 ps
5

b

1

32
65
131

0.8

0.8

265
538
EXC

0.6

0.6

0.4

0.4

0.2

0.2

0

0
550

600
650
Wavelength (nm)

700

550

15780

16150

d
SE Peak Frequency (cm-1)

SE Peak Frequency (cm-1)

700

c

Data
Fit

15760

15740

15720

15700

15680

600
650
Wavelength (nm)

0

100

200
300
Delay (ps)

400

500

16100

16050

16000

15950

15900

0

100

200
300
Delay (ps)

400

500

(
Figure(3.2.((SpectrallyVresolved!transient!grating!data!for!(a)!mPlum!and!the!(b)!
I65L!mutant!with!excitation!spectrum!(EXC)!overlaid.!!Center!peak!positions!for!the!
SE!band!obtained!from!transient!grating!data!for!(c)!mPlum!and!the!(c)!I65L!mutant!
(black!circles).!!A!bestVfit!curve!derived!from!nonlinear!least!squares!fitting!is!
overlaid!(solid!red!line).!!!
In!each!case,!we!observed!two!distinct!bands!corresponding!to!the!GSB!(high!
energy)!and!SE!(low!energy)!responses.!!The!absence!of!signal!between!bands!for!
mPlum!and!on!the!red!extreme!for!I65L!suggests!a!strong!excited!state!absorption!
(ESA).!!To!test!this!effect,!we!evaluated!our!signal!in!the!case!of!infinite!delay.!!For!a!
heterodyne!detected!signal!(e.g.#transient!absorption),!one!would!expect!the!signal!

80!
!
spectrum!upon!complete!relaxation!to!reflect!the!excitationVweighted!sum!of!steady!
state!absorption!and!emission.!!To!account!for!this!discrepancy!in!this!homodyne!
experiment,!the!SRTG!data!was!divided!by!the!probe!intensity!spectrum!as!
explained!previously!by!Lee!and!coworkers152.!!This!test!confirms!the!presence!of!
ESA!to!the!red!of!ωeg,!likely!overlapping!the!SE!band.!!Furthermore,!the!transient!
absorption!spectrum!of!mPlum!also!shows!a!negative!band!located!at!wavelengths!
both!redVshifted!and!blueVshifted!of!the!GSB!indicative!of!ESA.!!We!assume!the!ESA!
does!not!spectrally!evolve!on!the!experimental!timescale.!!!!
mPlum SRTA

1

0.8

∆T/T (a.u.)

0.6

0.4

0.2

0

−0.2

−0.4

520

540

560

580

600

620

Wavelength (nm)

640

660

680

700

(

Figure(3.3.!!SpectrallyVresolved!transient!absorption!spectrum!of!mPlum!(black)!
with!excitation!overlaid!(green).!!Negative!features!bounding!the!positive!bleach!
signal!reflect!the!presence!of!multiple!excited!state!absorption!(ESA)!contributions.!
!

Transient!grating!spectra!at!each!time!increment!were!fit!to!a!sum!of!two!

Gaussians!and!the!center!positions!of!each!band!were!subsequently!fit!to!a!
multiexponential!decay.!!Data!at!early!and!later!delays!were!omitted!due!to!finite!
pulse!effects!and!poor!signalVtoVnoise,!respectively.!!SE!peak!positions!and!fits!for!

81!
!
mPlum!and!I65L!are!displayed!in!Figure!3.2b!and!the!remaining!mutants!are!shown!
in!Figure!3.4.!!!

4

1.585

x 10

E16Q

4

1.64

x 10

E16H

4

1.61

x 10

E16L

1.58

1.575

SE Peak (cm−1)

SE Peak (cm−1)

SE Peak (cm−1)

1.608
1.635

1.63

1.625

1.606
1.604
1.602
1.6
1.598

1.57

0

200

1.62

400

0

200

T (ps)
4

1.615

x 10

T (ps)

I65A

I65V

0

200

1.596

0

50

100

150

T (ps)

1.585
SE Peak (cm−1)

SE Peak (cm−1)

x 10

4

1.59

1.61
1.605
1.6
1.595
1.59

400

1.58
1.575
1.57

0

200
T (ps)

400

1.565

400

T (ps)(

Figure(3.4.!!Stimulated!emission!(SE)!peak!positions!for!mPlum!mutants!(black!
circles).!!A!bestVfit!curve!derived!from!nonlinear!least!squares!fitting!is!overlaid!
(solid!red!line).!
The!fitting!results!and!associated!error!analysis!are!presented!in!Tables!3.3!and!3.4.!!!
(
(
(
(
(

82!
!
Table(3.3.!!Summary!of!SRTG!fitting!results!following!fitting!the!raw!data!to!a!sum!
of!two!Gaussians.!!!
Mutant&SE&Red&Shift&
Shift&
Tau&(ps)&
Magnitude&
41(cm )&
3&
17&&&
mPlum&

GSB&Blue&Shift&
Shift&
Tau&(ps)&
Magnitude&
41(cm )&
44&
44&

51&

95&

58&

46&

E16Q&

107&

26&

86&

21&

E16H&

49&

69&

44&

44&

E16L&

13&

23&

34&

7&

I65L&

44&

24&

237&

19&

I65A&

44&

44&

9&

16&

&&

100&

37&

173&

29&

I65V&

2&

19&

19&

13&

51&

84&

142&

18&

!
Table(3.4.!!Fitting!error!analysis!for!multiexponential!fits!to!SE!band!shifts.!!The!
given!values!represent!95%!confidence!intervals!for!the!parameters!of!the!fit!
equation:! f (t ) = a ⋅ e−bt + c ⋅ e−dt .!!Amplitudes!are!expressed!in!wavenumbers!(cmV1)!
and!time!constants!are!reported!as!eV1!lifetimes!in!picoseconds.!!!!
Mutant&41

a&(cm )&

41

b&(ps)&

c&(cm )&

d&(ps)&

mPlum&

11.1&

22.1&

2&

14&

89.4&

100.5&

46&

57&

E16Q&

24.5&

26.6&

93&

128&

44&

44&

44&

44&

E16H&

67.3&

71.6&

44&

54&

44&

44&

44&

44&

E16L&

20.2&

24.8&

10&

18&

44&

44&

44&

44&

I65L&

20.7&

28.0&

27&

120&

44&

44&

44&

44&

I65A&

27.4&

31.2&

62&

103&

44&

44&

44&

44&

I65V&

12.5&

25.6&

1&

17&

77.6&

89.4&

45&

60&

!
!

The!solvation!responses!can!be!categorized!in!terms!of!either!timescale!or!

amplitude.!!Timescales!ranged!from!around!a!hundred!ps!(E16Q,!I65A),!tens!of!ps!
(mPlum,!E16H,!E16L,!I65L),!and!a!few!ps!(mPlum,!I65V).!!It!should!be!noted!that!the!
mPlum!time!constants!of!3!and!51!ps!are!consistent!with!those!measured!with!
fluorescence!upconversion!by!Abbyad!et#al.!(4.3!and!71!ps)45.!!The!shift!magnitudes!
fell!into!two!groups:!22V37!cmV1!(E16Q,!E16L,!I65L,!I65A)!and!65V112!cmV1!(mPlum,!

83!
!
I65V,!E16H).!!The!mutants!with!the!largest!Stokes!shifts!(mPlum,!I65V)!have!
multiple!timescales!of!relaxation.!!The!remaining!show!only!a!single!timescale!of!
tens!of!ps.!!Longer!decay!times!cannot!be!ruled!out!given!the!500!ps!experimental!
time!window.!!Timescales!of!the!GSB!component!are!in!qualitative!agreement!
suggesting!similar!ground!and!excited!state!dynamics!in!accordance!with!a!linear!
response.!!The!magnitudes!of!the!spectral!shifts!in!all!cases!are!smaller!than!
observed!by!Abbyad!et#al.!(793!cmV1).!!In!principle,!this!can!be!attributed!to!either!
inadequate!time!or!spectral!resolution.!!The!former!is!unlikely!since!the!time!
resolution!in!the!previous!study!was!on!the!order!of!100s!of!fs,!whereas!SRTG!was!
performed!with!20!fs!excitation!pulses.!!On!the!other!hand,!reductions!in!the!
measured!spectral!shifts!are!inevitable!given!a!finite!excitation!bandwidth!of!
approximately!1000!cmV1!along!with!an!overlapping!ESA.!!
!

The!SRTG!response!was!modeled!to!reconcile!the!disparity!in!shift!magnitude!

between!these!measurements!and!upconversion!spectroscopy.!!We!consider!the!
influence!of!a!finite!laser!bandwidth!and!ESA!on!the!SE!band!position.!!For!three!
electronic!levels,!there!are!three!possible!resonant!time!ordered!contributions!to!
the!third!order!optical!response!to!consider!for!this!experimental!phaseVmatching!
direction158.!!To!simplify!fitting,!the!GSB!response!was!omitted.!!This!simplification!
will!not!affect!the!results!since!this!band!is!blueVshifted!relative!to!the!SE!band.!!An!
initial!center!position!of!16178!cmV1!was!chosen!for!the!SE!component!with!an!
asymmetric!Gaussian!shape!replicating!the!steady!state!emission!spectrum.!!The!
excitation!pulse!center!and!width!mimicked!that!used!in!the!experiment!(16000!cmV
1,!1025!cmV1).!!An!asymmetric!Gaussian!with!a!shape!closely!matched!to!the!ground!

84!
!
state!absorption!spectra!was!used!to!model!ESA.!!The!intensity!of!the!ESA!transition!
was!assumed!equal!to!0.4!of!the!GSB!consistent!and!centered!at!16667!cmV1,!
consistent!with!the!transient!absorption!spectrum.!!The!SE!and!ESA!signal!
responses!were!added!according!to!the!signs!of!their!respective!optical!responses.!!
A!red!shift!was!assumed!to!follow!the!measured!upconversion!time!constants!and!
shift!magnitudes!for!mPlum!of!4.3!(301!cmV1)!and!71!ps!(492!cmV1).!!Figure!3.5!
illustrates!the!modeled!data!with!and!without!application!of!a!finite!excitation!
bandwidth!and!ESA.!!Inclusion!of!these!effects!yields!a!total!shift!magnitude!of!137!
cmV1,!in!close!agreement!with!our!experimentally!measured!value.!!Moreover,!the!
shift!time!constants!of!5!and!72!ps!for!the!filtered!signal!closely!match!the!inputted!
values.!!By!proportionally!decreasing!the!shift!magnitude!relative!to!mPlum!in!Table!
3.2,!we!also!find!the!calculated!shift!magnitudes!similarly!agree!with!the!
experimental!values!for!the!mutants.!!This!analysis!demonstrates!that!accounting!
for!a!finite!excitation!bandwidth!and!ESA!explains!the!decreased!shift!magnitudes!
relative!to!upconversion!spectroscopy!and!that!SRTG!is!sensitive!to!the!underlying!
constants!of!the!excited!state!reorganization.!!!

85!
!
1.2

a

EXC
ESA

Normalized Amplitude (a.u.)

Normalized Amplitude (a.u.)

1.2
1
0.8
0.6
0.4
0.2
0
1.4

1.5

1.6

1.7
ï

Frequency (cm )

b

EXC
ESA

1
0.8
0.6
0.4
0.2
0
1.4

1.8

1.5

1.6

1.7
ï

4

Frequency (cm )

x 10

1.8
4

x 10

Figure(3.5.((Simulated!SRTG!data!with!and!without!application!of!a!finite!excitation!
bandwidth!(EXC)!and!ESA!response.!!(a)!Hypothetical!SRTG!data!without!excitation!
filter!and!ESA.!!(b)!With!excitation!filter!and!ESA.!!
The!amplitude!decays!of!each!band!reveal!predictable!photophysical!
behavior!(Table!3.5).!
Table(3.5.!!SRTG!amplitude!decays!of!GSB!and!SE!bands.!
Mutant&SE&&&

Tau&(ps)&

Amplitude&

Tau&(ps)&

Amplitude&

mPlum&

22&

0.29&

52&

0.21&

325&

0.71&

434&

0.79&

98&

0.30&

152&

0.22&

E16Q&

498&

0.67&

533&

0.78&

E16H&

443&

1.00&

422&

1.00&

E16L&

58&

0.13&

494&

0.99&

549&

0.85&

44&

44&

445&

0.97&

211&

0.26&

44&

44&

581&

0.71&

266&

0.10&

384&

0.87&

437&

0.84&

44&

44&

19&

0.16&

19&

0.10&

360&

0.81&

394&

0.90&

I65L&
I65A&
I65V&

!

GSB&

!

86!
!
In!the!case!of!SE,!the!dominant!component!closely!matches!the!measured!excited!
state!lifetime!of!800!ps!for!mPlum!after!correcting!twofold!due!to!the!effect!of!
homodyne!detection45.!!In!some!mutants,!minor!contributions!exist!on!both!shorter!
and!longer!timescales!consistent!with!complex!photophysics!known!to!RFPs.!!The!
GSB!band!intensity!decay!reflects!the!ground!state!recovery!process,!which!is!
expected!to!follow!both!directly!from!the!excited!state,!or!more!slowly!from!a!third!
level,!as!RFPs!are!known!to!undergo!varying!degrees!of!dark!state!conversion!with!
repopulation!occurring!on!microsecond!and!longer!timescales146,159.!!!This!effect!
would!yield!a!disparity!between!rates!of!ground!state!repopulation!and!excited!state!
depopulation.!!The!amplitude!decays!are!also!slightly!influenced!by!bands!shifting!
within!the!finite!laser!bandwidth.!!We!will!not!further!consider!the!GSB!recovery!
timescales,!but!instead!focus!on!the!excited!state!dynamics!revealed!by!the!
timescales!of!SE!band!shifts.!
Molecular#Dynamics#Simulations#
MD!trajectories!of!each!mutant!were!used!to!examine!the!chromophore!
flexibility!and!the!interconversion!between!hydrogen!bond!states.!!First!we!
examined!the!dynamics!of!the!interaction!between!positions!16!and!65!in!mPlum!
and!found!two!distinct!conformations,!displayed!in!Figure!3.6.!!The!important!
difference!between!Figure!3.6!(a)!and!(b)!is!the!hydroxyl!group!orientation!(OE2!
and!HE2!in!the!CHARMM!file)!of!E16.!!In!Figure!3.6a,!the!OH!group!is!positioned!so!
that!it!can!make!a!direct!hydrogen!bond!with!I65,!whereas!in!Figure!3.6b,!it!
reorients!to!accommodate!a!waterVmediated!hydrogen!bond.!!The!simulations!

87!
!
reveal!the!presence!of!approximately!5!water!molecules!within!3!Å!of!the!
chromophore!for!all!mutants!(Figure!3.7).!!!!!!!

!
Figure(3.6.((Unique!16V65!hydrogen!bond!conformations!identified!in!MD!
simulations!of!mPlum.!!(a)!The!E16!hydroxyl!group!orientation!provides!a!small!
enough!separation!from!I65!to!allow!a!direct!hydrogen!bond.!!(b)!The!E16!hydroxyl!
group!orientation!results!in!a!larger!separation!from!I65!and!a!waterVmediated!
hydrogen!bond.!

88!
!

(
Figure(3.7.((Water!occupancy!within!3!Å!of!chromophore!over!a!40!ns!time!
trajectory!for!mPlum!and!variants.!!!
In!the!mPlum!variants,!we!found!that!some!can!also!form!two!distinct!16V65!
hydrogen!bonds!(E16Q,!I65V),!whereas!other!mutants!can!only!form!one!type!of!
hydrogen!bond!(I65A,!I65L,!E16H)!or!none!at!all!(E16L).!!Figure!3.8!presents!times!
series!trajectories!from!MD!simulations!from!0V40!ns.!!The!relative!16V65!
positioning!is!expressed!as!Δr,!and!is!defined!as!the!distance!between!the!
participating!atom!in!the!16!sidechain!and!the!NVacylimine!oxygen!of!residue!65.!!!

89!
!

!
Figure(3.8.((MD!time!series!trajectories!for!all!mPlum!mutants.!!Δr!refers!to!the!
distance!between!the!sidechain!of!16!and!the!NVacylimine!oxygen!of!I65.!
The!capability!to!switch!between!substates!is!correlated!with!Stokes!shift.!!
For!the!three!proteins!with!the!largest!Stokes!shift,!mPlum,!E16Q,!and!I65V,!there!
are!distinct,!nsVtimescale!periods!in!which!the!separation!is!characteristic!of!
relatively!stable!direct!hydrogen!bonds!(<3.5!Å),!as!well!as!switching!to!distinct!
periods!in!which!the!separation!is!characteristic!of!waterVmediated!hydrogen!bonds!

90!
!
(>4!Å).!!This!is!reflected!in!the!first!two!columns!of!Table!3.4,!which!shows!that!for!
these!proteins,!conformational!interconversion!allows!each!type!of!hydrogen!bond!
to!occur!for!a!significant!fraction!of!the!MD!simulation.!!For!the!other!four!mutants,!
with!smaller!Stokes!shifts,!the!time!series!trajectories!of!Figure!3.8!do!not!reveal!
conversion!between!two!relatively!stable!types!of!hydrogen!bonds.!!Mutants!I65L!
and!I65A!spend!most!of!their!trajectories!in!a!direct!hydrogen!bond,!while!E16H!
fluctuates!primarily!within!the!waterVmediated!hydrogen!bond!conformation.!!
Finally,!the!E16L!variant!cannot!form!a!stable!hydrogen!bond!of!either!variety!due!
to!its!aliphatic!nature!and!hence,!exhibits!random!fluctuations!throughout!the!
trajectory.!!!

!
Figure(3.9.((MD!time!series!trajectory!for!the!mPlum!E16L!mutant.!!Δr!refers!to!the!
distance!between!the!L16!sidechain!and!the!NVacylimine!oxygen!of!I65.!
The!16V15!distance!for!E16Q!shows!an!additional!substate!with!Δr!~!6.5!Å,!caused!
by!a!unique!rotation!of!Q16.!!A!histogram!of!the!16V65!distance!r!for!each!FP!

91!
!
similarly!reveals!the!presence!of!these!distinct!hydrogen!bond!configurations!
described!above!(Figure!3.10).!

ab!
Figure(3.10.!!Histograms!of!the!probability!for!a!16V65!separation,!Δr,!to!occur!
during!the!MD!simulations.!!(a)!The!three!mutants!with!the!largest!Stokes!shift!show!
two!distinct,!stable!configurations,!corresponding!to!Δr!characteristic!of!a!direct!
hydrogen!bond!as!well!as!a!larger!Δr!characteristic!of!a!waterVmediated!hydrogen!
bond.!!(b)!The!four!mutants!with!the!smaller!Stokes!shift!display!a!single!peak!
implying!little!or!no!fluctuations!in!16V65!hydrogen!bond.!
Table(3.6.((Survival!times!calculated!for!the!direct!hydrogen!bond!molecular!
configuration!and!for!the!waterVmediated!hydrogen!bond!configurations!following!
two!hydrogen!bond!criteria.!!The!survival!times!listed!are!obtained!from!the!single!
exponential!fits!to!the!time!correlation!function.!!Calculation!of!survival!times!was!
not!possible!for!cases!with!negligible!hydrogen!bond!percentage.!!Survival!times!are!
given!using!two!different!sets!of!criteria!for!determining!hydrogen!bonds!(angle!cutV
off!of!30°!versus!40°!in!VMD).!
Mutant&!

Survival&Time&(ps)&3.5&Å/30°&

Survival&Time&(ps)&3.5&Å/40°&&&&Direct&

H2O&
mediated&

Direct&H2O&
mediated&

mPlum&

10&

2&

29&

4&

E16Q&

3&

2&

5&

3&

E16H&

44&

2&

44&

4&

E16L&

44&

44&

44&

44&

I65L&

13&

44&

44&

44&

I65A&

29&

44&

167&

44&

I65V&

8&

2&

17&

3&

92!
!
We!next!analyzed!the!MD!results!to!determine!if!the!lifetimes!of!the!various!
hydrogen!bond!configurations!are!consistent!with!their!characteristic!experimental!
timescales.!!Simulations!were!carried!out!for!time!intervals!long!enough!to!ensure!
exhaustive!sampling!of!all!molecular!subconformations160.!!We!extracted!hydrogen!
bond!survival!times!from!the!simulations!by!calculating!the!hydrogen!bond!survival!
function!in!the!direct!substate,!and!separately!in!the!waterVmediated!substate.!!The!
survival!function!gives!the!conditional!probability!that!a!hydrogen!bond!intact!at!
time!!!!! !will!also!remain!intact!at!time!!! !and!is!given!by p(t ) = 1 − f (t ) ,!where!f(t)!is!
the!fraction!of!hydrogen!bonds!broken!at!time!t.!!The!average!survival!time!was!
estimated!by!fitting!the!survival!function!to!an!exponential:! p(t ) ~ exp(− t τ )!161.!!The!
survival!time!functions!show!decays!on!a!few!ps!and!tens!of!ps!timescales!for!most!
mutants.!!!

93!
!

&
Figure(3.11.!!Survival!functions!calculated!from!the!MD!simulations!for!the!direct!
and!waterVmediated!hydrogen!bonds!(3.5!Å/30°!cutVoff).!!
Both!sets!of!decays!show!qualitative!agreement!with!the!experimental!time!
constants!shown!in!Table!3.6,!with!the!30°!criteria!being!proportionally!shorter!for!
the!direct!hydrogen!bond.!!For!variants!with!significant!populations!in!both!
substates,!it!is!observed!that!direct!hydrogen!bonds!survive!for!much!longer!times!
than!do!the!waterVmediated!hydrogen!bonds.!!This!implies!that!the!shorter!

94!
!
experimental!time!constants!may!be!due!to!16V65!waterVmediated!hydrogen!bond!
dynamics,!whereas!the!longer!experimental!time!constants!may!be!due!to!16V65!
direct!hydrogen!bond!dynamics.!!If!the!percentage!of!a!type!of!hydrogen!bond!is!
very!small,!we!are!not!able!to!calculate!survival!times,!which!might!explain!why!
mutants!of!smaller!Stokes!shift!have!only!one!experimentally!observed!time!
constant.!!In!general,!the!biggest!disagreements!fall!among!the!E16!mutants.!!The!
absence!of!shorter!time!component!in!E16Q!is!likely!a!result!of!poor!signalVtoVnoise!
given!its!comparably!smaller!shift!magnitude.!!A!strong!ESA!overlap!with!the!SE!
band!would!have!an!effect!similar!to!a!finite!excitation!bandwidth!mentioned!above!
in!that!it!would!decrease!the!magnitudes!of!the!spectral!shifts.!!The!longer!time!
constant!observed!in!this!mutant!can!be!explained!in!terms!of!a!unique!rotation!of!
Q16!with!a!calculated!correlation!time!of!91!ps,!which!closely!matches!the!
experimental!value!of!112!ps.!!For!E16L!and!E16H,!the!origin!of!this!discrepancy!is!
less!obvious,!although!we!find!an!elevated!chance!of!a!waterVI65!hydrogen!bond!
(without!a!simultaneous!waterV16!hydrogen!bond)!in!these!mutants!(Table!3.7).!!!
Table(3.7.((Probability!of!I65Vwater!hydrogen!bond!without!a!simultaneous!16V
water!hydrogen!bond!from!the!MD!simulation!for!each!mutant.!

!

Mutant&H204I65&(%)&

mPlum&

12.8&

E16Q&

30.5&

E16H&

30.8&

E16L&

49.1&

I65L&

13.1&

I65A&

3.4&

I65V&

32.6&

95!
!
The!survival!time!of!this!hydrogen!bond!is!only!a!few!picoseconds,!and!therefore!the!
observed!relaxation!is!clearly!not!the!result!of!the!dynamics!of!this!bond.!!It!is!
possible!that!water!motion!in!this!region!near!the!chromophore!could!facilitate!
solvation!on!the!timescales!observed.!!In!general,!the!mutations!at!position!16!yield!
less!predictable!responses!than!those!at!position!65,!probably!because!these!
mutations!replace!a!charged!side!chain!with!uncharged,!polar!or!nonpolar!
sidechains,!which!are!likely!induce!greater!electrostatic!perturbation!of!the!
chromophore!environment!versus!the!position!65!mutations.!!Further!analysis!will!
require!quantum!mechanical!calculations.!!!
To!connect!the!dynamics!observed!in!the!MD!trajectories!with!Stokes!shift,!
we!examined!the!influence!of!the!16V65!interaction!on!hydrogen!bonds!made!to!the!
chromophore!with!the!surrounding!environment.!!In!particular,!interactions!with!
S146,!R95,!E215,!and!Q109!are!known!to!be!important!for!spectral!tuning!in!other!
green!and!red!FPs11.!!Table!3.8!summarizes!this!analysis.!!The!first!two!columns!
show!the!fraction!of!time!spent!hydrogenVbonded,!direct!or!waterVmediated,!at!
positions!16!and!65.!!The!remaining!columns!tabulate!the!time!a!chromophoreV
sidechain!hydrogen!bond!occurs,!partitioned!by!16V65!substate,!and!normalized!by!
its!respective!occupancy.!!Residue!65!is!considered!to!be!part!of!the!extended!
chromophore!and!therefore!hydrogen!bonding!with!65!can!directly!influence!the!
chromophore!environment10.!!ChromophoreVhydrogen!bond!interactions!at!other!
sidechains!appear!independent!of!whether!there!is!a!direct!or!waterVmediated!
interaction!at!16V65.!!We!conclude!that!16V65!switching!constitutes!the!largest!

96!
!
observable!structural!change!within!the!chromophore!environment!and!must!be!
integral!for!the!bathochromic!shift!observed!in!mPlum.!!!
Table(3.8.!!Hydrogen!bonds!to!chromophore!from!neighboring!sidechains!are!
presented!in!terms!of!time!fraction!as!calculated!from!MD!simulations.!!For!each!
mutant,!bonds!with!sidechains!S146,!R95,!E215,!and!Q109!are!reported!for!each!16V
65!substate,!direct!and!H OVmediated,!normalized!by!their!respective!occupancy.!!
For!all!entries,!3.5!Å!distance!and!30°!angle!cutVoff!values!were!used!for!determining!
the!hydrogen!bonds.!
2

Mutant&16465&H4Bond&(%)&

S1464CRO&(%)&

R954CRO&(%)&

E2154CRO&(%)&

Q1094CRO&(%)&&&&Direct&

H2O&
mediated&&Direct&

H2O&
mediated&&Direct&

H2O&
mediated&

Direct&H2O&
mediated&&Direct&

H2O&
mediated&

mPlum&

31.3&

18.4&

72.8&

49.9&

88.9&

89.7&

82.3&

79.6&

38.7&

10.9&

E16Q&

18.4&

14.3&

80.6&

85.9&

93.4&

95.1&

79.3&

80.1&

43.6&

58.5&

E16H&

0.0&

25.2&

85.7&

43.6&

85.7&

87.2&

71.4&

80.9&

28.6&

6.2&

E16L&

0.3&

0.2&

84.0&

93.0&

94.0&

84.0&

74.5&

66.6&

20.3&

26.7&

I65L&

76.8&

0.0&

3.0&

0.0&

85.8&

83.3&

86.5&

83.3&

34.9&

50.0&

I65A&

89.9&

0.5&

83.5&

95.6&

91.9&

89.1&

86.4&

86.9&

58.7&

45.7&

I65V&

28.2&

14.8&

27.6&

38.9&

64.9&

66.6&

82.2&

81.8&

11.1&

6.3&

(
The!chromophore!flexibility!was!analyzed!by!extracting!a!root!mean!square!
fluctuation!(RMSF)!on!an!atomic!basis!from!the!MD!simulations.!!We!observe!no!
correlation!between!chromophore!flexibility!and!Stokes!shift!among!these!mutants!
(Figure!3.12).!!!

97!
!

(
Figure(3.12.!!Calculated!chromophore!RMSF!for!each!mutant.!!
(
3.4(Discussion(
The!time!dependent!shifts!of!the!SE!band!observed!in!SRTG!measurements!
are!directly!related!to!the!relaxation!of!the!excited!state!energy!leading!to!the!Stokes!
shift!of!the!steady!state!emission!spectrum.!!Classical!MD!simulations!are!not!
capable!of!estimating!the!magnitude!of!electronic!energy!relaxation!since!they!only!
model!dynamics!on!the!ground!electronic!state.!!However,!within!linear!response!
theory,!these!dynamics!approximate!motions!leading!to!excited!state!solvation!and!
thus,!serve!as!a!means!of!modeling!the!effect!of!variations!in!chromophoreV
environment!interactions!on!molecular!dynamics!and!their!timescales162.!!These!
results!provide!new!insight!into!the!dynamics!of!the!interaction!between!the!16!and!
65!sidechains!in!mPlum!and!an!explanation!for!its!large!Stokes!shift!relative!to!other!
DsRedVderived!FPs.!!These!conclusions!follow!from!two!key!observations.!!First,!the!
two!timescales!observed!experimentally!and!in!the!simulations!represent!survival!
times!of!direct!and!waterVmediated!hydrogen!bond!substates!between!the!16!and!

98!
!
65!sidechains.!!Second,!there!is!a!direct!correlation!between!the!Stokes!shift!of!each!
mutant!and!to!the!extent!to!which!it!undergoes!hydrogen!bond!interconversion.!!
Cases!in!which!no!direct!hydrogen!bond!is!possible!(E16L,!E16H)!or!where!a!direct!
hydrogen!bond!forms!almost!exclusively!(I65L,!I65A)!show!the!smallest!Stokes!
shifts!of!mutants!tested.!!Conversely,!species!with!the!greatest!propensity!to!
interconvert!between!hydrogen!bond!states!(mPlum,!E16Q,!and!I65V)!have!the!
largest!Stokes!shift.!!This!dynamic!model!of!the!chromophore!solvation!stands!in!
contrast!with!previous!studies!of!mPlum!mentioned!above,!which!primarily!
attributed!its!redVshifted!emission!to!stabilization!of!the!excited!state!through!a!
direct!hydrogen!bond!between!E16!and!the!NVacylimine!oxygen!of!I65.!!!
The!influence!of!confined!water!molecules!on!the!solvation!of!probes!buried!
within!protein!environments!following!photoexcitation!has!brought!about!intense!
debate163,164.!!In!particular,!it!is!questioned!whether!confined!water!or!protein!
sidechains!are!responsible!for!the!slow!(relative!to!bulk!water)!ps!solvation!
observed!in!these!environments.!!For!GFP,!Xu!and!coworkers!found!10!water!
molecules!in!close!vicinity!to!the!chromophore!with!predicted!solvation!dynamics!
timescales!ranging!from!tens!of!fs!to!several!hundred!ps165.!!While!the!simulations!
indicate!a!similar!presence!of!water,!it!is!unlikely!that!these!solvent!molecules!are!
responsible!for!the!variation!in!dynamics.!!First,!we!observe!similar!water!
occupancy!among!all!variants!indicating!any!contribution!is!likely!consistent!
regardless!of!mutation!(Figure!3.7).!!Moreover,!we!do!not!observe!the!slowest!333!
ps!time!constant!predicted!by!Xu!et#al.!in!any!sample.!!It!is!possible!that!the!
anomalous!responses!of!the!E16!mutants,!noted!above,!is!related!to!a!waterVI65!

99!
!
hydrogen!bond!not!observed!in!the!I65!variants.!!These!observations!suggest!water!
may!play!a!minor!role!in!the!observable!dynamic!Stokes!shift,!but!the!dominant!
response!is!governed!by!internal!hydrogen!bond!dynamics!as!reflected!by!the!above!
survival!time!calculations!for!most!species.!!
It!is!clear!that!the!presence!of!neither!a!direct!or!waterVmediated!hydrogen!
bond!is!the!sole!determinant!of!a!large!Stokes!shift.!!It!is!possible!that!the!16V65!
interaction!modulates!chromophore!flexibility.!!Flexibility!about!the!methylidene!
bridge!would!disrupt!coplanarity!between!the!imidazole!and!phenoxy!moieties,!
inhibit!electron!delocalization,!and!lead!to!a!smaller!Stokes!shift19,!47.!!A!similar!effect!
was!proposed!in!quantum!mechanical!simulations!of!DsRed!where!a!noted!torsion!
of!the!terminal!carbonyl!of!I65!leads!to!a!blue!shift!due!to!loss!of!conjugation!as!
observed!in!mOrange11,!148.!!It!is!plausible!hydrogen!bonding!to!this!position!serves!
as!an!anchor!that!preserves!coplanarity!either!at!the!carbonyl!or!more!broadly!
across!the!ring!system.!!Among!the!series!of!mutants,!this!may!be!manifest!in!
slightly!different!ways!given!the!precise!nature!of!the!16V65!interaction.!!These!can!
be!broken!down!into!four!cases:!!1.!forms!no!hydrogen!bond!(E16L),!2.!forms!a!
direct!hydrogen!bond!(I65A,!I65L),!3.!forms!a!waterVmediated!hydrogen!bond!
(E16H),!4.!demonstrates!interconversion!between!multiple!hydrogen!bond!states!
(mPlum,!E16Q,!I65V).!!Assuming!E16L!as!a!baseline,!those!with!a!single!interaction!
(I65A,!I65L,!and!E16H)!represent!the!middle!grouping!of!observed!Stokes!shifts,!
whereas!those!in!the!fourth!category!(mPlum,!E16Q,!I65V)!hold!the!largest!Stokes!
shifts.!!The!influence!of!switching!is!less!obvious,!but!it!may!be!true!that,!on!average,!
coplanarity!is!higher!among!those!interconverting!between!conformations.!!!

100!
!
Alternatively,!the!most!coplanar!configuration!(local!energetic!minimum)!may!lie!
along!the!switching!coordinate!and!remain!unpopulated!by!those!not!able!to!
interconvert.!!Lastly,!switching!between!states!may!act!to!suppress!flexibility!of!the!
chromophore!in!dimensions!other!than!ring!torsion,!which!would!also!help!drive!
stabilization!of!the!excited!state.!!It!is!interesting!to!note!that!of!the!I65!mutants,!
only!the!valine!derivative!undergoes!hydrogen!bond!switching,!while!I65A!and!I65L!
demonstrate!more!rigid!conformations.!!We!suggest!this!may!be!a!steric!effect!and!
possibly!essential!to!controlling!hydrogen!bond!interconversion.!!
ChromophoreVsidechain!hydrogen!bonding!interactions!are!observed!in!
crystal!structures!of!mCherry,!mStrawberry,!mNeptune,!mRojoA,!mRouge,!eqFP650,!
and!eqFP67011,!18,!47,!49.!!These!findings!highlight!the!importance!of!the!dynamics,!not!
the!mere!presence!of!these!interactions,!and!suggest!that!Stokes!shift!is!related!to!
the!capability!of!interconversion!between!multiple!hydrogenVbonded!states.!!It!
seems!reasonable!to!hypothesize!that!this!behavior!scales!with!the!number!of!weak,!
interconvertible!chromophore!hydrogen!bond!interactions.!!Consistent!with!this!
idea,!a!recent!publication!describing!the!structure!and!properties!of!the!farthest!red!
emitting!FP!engineered!to!date,!TagRFP675,!which!has!a!75!nm!Stokes!shift50.!!They!
attribute!its!red!emission!to!several!hydrogen!bonding!contacts!involving!Q41!and!
S28!at!the!NVacylimine!position!with!additional!hydrogen!bonds!at!the!phenoxy!end!
of!the!chromophore!with!N143,!N158,!and!R197.!!
These!results!have!significant!implications!in!the!context!of!engineering!an!
ideal!red!fluorescent!protein,!i.e.!one!with!both!redVshifted!emission!and!high!

101!
!
quantum!yield!desired!for!cellular!imaging!applications.!!Previous!studies!
recognized!an!apparent!tradeoff!between!these!properties,!with!brightness!
hindered!by!fast!nonradiative!deactivation,!whereas!redVshifted!emission!results!
from!environmental!solvation!of!the!excited!state19.!!The!implication!is!that!
chromophores!of!the!brightest!species,!with!the!smallest!Stokes!shifts,!are!contained!
within!rigid!environments!unable!to!deactivate!excitation!through!thermal!
fluctuations,!phenolic!rotation,!or!cisVtrans!isomerization,!while!the!reddest!
emission!requires!local!flexibility!to!facilitate!solvation!of!the!excited!state!
electronic!distribution.!!We!hypothesize!that!the!hydrogen!bond!switching!
phenomenon!observed!here!acts!to!selectively!tether!the!chromophore!in!such!a!
manner!that!provides!a!degree!of!transient!flexibility!combined!with!stabilization!of!
the!excited!state.!!A!similar!effect!is!observed!in!mNeptune!where!the!guanidinium!
group!of!R197!runs!parallel!along!the!chromophore!and!is!thought!to!provide!
stability!across!the!ring!system47.!!This!“transient!flexibility”!may!provide!a!
mechanism!for!simultaneous!optimization!of!these!two!photophysical!properties.!!!!
3.5(Conclusions(
TimeVresolved!spectroscopy!was!carried!out!on!mPlum!and!a!panel!of!
strategic!point!mutants.!!The!experimental!results,!supported!by!MD!simulations,!
provide!new!insight!into!the!dynamics!of!the!interaction!between!the!16!and!65!
sidechains!in!mPlum!and!a!possible!explanation!for!its!large!Stokes!shift!relative!to!
other!DsRedVderived!FPs.!!These!conclusions!arise!following!two!key!observations.!!
First,!the!two!timescales!observed!experimentally!and!in!the!simulations!represent!
interconversion!between!direct!and!waterVmediated!hydrogen!bonding!substates!

102!
!
between!the!16!and!65!sidechains.!!Second,!there!is!a!direct!correlation!between!the!
Stokes!shift!of!each!mutant!and!to!the!extent!to!which!it!undergoes!hydrogen!bond!
interconversion.!!Cases!in!which!no!direct!hydrogen!bond!is!possible!(E16L,!E16H)!
or!where!a!direct!hydrogen!bond!forms!almost!exclusively!(I65L,!I65A)!show!the!
smallest!Stokes!shifts!of!mutants!tested.!!Conversely,!species!with!the!greatest!
propensity!to!interconvert!between!hydrogen!bond!states!(mPlum,!E16Q,!and!I65V)!
have!the!largest!Stokes!shift.!!This!dynamic!model!of!the!chromophore!stands!in!
contrast!with!previous!studies!of!mPlum,!which!primarily!attributed!its!redVshifted!
emission!to!direct!stabilization!of!the!excited!state!through!a!hydrogen!bond!
between!E16!and!the!NVacylimine!oxygen!of!I65.!!We!hypothesize!such!dynamics!
facilitate!a!bathochromic!shift!through!a!combination!of!direct!excited!state!
solvation!and!regulation!of!chromophore!flexibility.!!This!model!may!provide!a!
guide!for!further!development!of!highly!desirable!redVemitting!FPs.!!Moreover,!these!
findings!demonstrate!the!utility!of!ultrafast!spectroscopy!in!measuring!functionally!
relevant!sidechain!fluctuations,!not!typically!resolvable!with!steady!state!methods.!(
(
!
!
!
!
!
!
!
!

103!
!
Chapter(IV(
The(Origin(of(Extended(Stokes(Shift(of(TagRFP675((
4.1(Introduction(
Modification!of!the!progenitor!eqFP578,!originally!extracted!from!the!sea!
anemone!Entacmaea#quadricolor,!led!to!the!development!of!a!redVemitting!
monomeric!homologue!mKate12.!!The!nearVinfrared!fluorescent!proteins!TagRFP675!
and!TagRFP657!emerged!from!directed!evolution!of!mKate!and!represent!the!
reddestVemitting!members!among!all!GFPVlike!FPs!reported!to!date12,!50,!166.!!
TagRFP675!exhibits!a!Stokes!shift,!which!is!30!nm!redVshifted!comparing!to!that!of!
its!parental!protein.!!Its!extended!Stokes!shift!was!suggested!to!be!due!to!an!
extensive!hydrogenVbonding!network!between!the!chromophore!and!its!protein!
matrix.!!Indeed,!as!revealed!by!highVresolution!crystal!structures!of!TagRFP675,!its!
DsRedVlike!chromophore!forms!multiple!direct!and!waterVmediated!hydrogenV
bonding!interactions!with!the!sidechains!of!S28,!Q41,!Q106,!N143,!N158,!and!R197.!!
In!particular,!interactions!involving!the!NVacylimine!carbonyl!of!DsRedVlike!
chromophores!appear!to!have!a!strong!influence!on!excited!state!properties!of!FPs44.!!
In!TagRFP675,!the!NVacylimine!carbonyl!forms!two!hydrogen!bonds.!!One!arises!
from!a!direct!interaction!with!the!Q41!sidechain,!while!the!other!stems!from!an!
embedded!water!molecule!supported!by!tertiary!interactions!with!amide!group!of!
Q106.!!These!interactions!are!illustrated!in!Figure!4.1.!!A!similar!motif,!formed!by!
side!chains!of!Q41!and!T28,!is!observed!in!another!mKate!derivative,!mCardinal167.!!
This!interaction!was!suggested!to!provide!enhanced!excited!state!stabilization!
through!a!stronger!direct!hydrogen!bond!interaction167.!!A!waterVmediated!

104!
!
hydrogen!bond!between!the!chromophore!and!S28!located!on!the!inner!wall!of!the!
βVbarrel!was!realized!in!the!mCardinal!precursor,!named!mNeptune47.!!
We!previously!investigated!a!similar!interaction!between!the!NVacylimine!
carbonyl!of!the!chromophore!and!carboxylate!group!of!the!E16!residue!in!mPlum45V
46.!!Utilizing!timeVresolved!spectroscopy!and!supported!by!Molecular!Dynamics!

(MD)!simulations,!we!discovered!that!redVshifted!emission!correlates!not!with!
hydrogen!bond!strength,!but!a!facile!switching!between!direct!and!waterVmediated!
hydrogen!bond!conformers168.!!This!model!suggests!that!localized!flexibility!may!be!
important!to!excited!state!stabilization.!!

Q106

Chromophore

S28

Q41

(
Figure(4.1.!The!hydrogen!bond!interactions!between!the!NVacylimine!oxygen!of!the!
DsRedVlike!chromophore,!S28,!Q41,!Q106!side!chains!and!water!molecule!identified!
in!the!crystal!structure!of!TagRFP675!(pdb!code!4KGF).!!Hydrogen!bonds!are!
represented!as!dashed!black!lines;!atoms!are!colored!by!atom!type;!water!molecule!
is!shown!as!red!sphere.!

105!
!
The!molecular!response!in!the!condensed!phase!can!be!probed!with!timeV
resolved!electronic!spectroscopy.!!The!excited!chromophore!is!coupled!to!stochastic!
fluctuations!of!the!surrounding!bath!with!a!spectral!density!spanning!an!
extraordinary!wide!frequency!range!encompassing!ultrafast!sidechain!motions,!
intramolecular!vibrations,!or!tertiary!interactions!to!much!slower!global!structural!
rearrangements!(>100s!of!ps)!or!even!discrete!conformational!states!(isomers)59.!!
Such!dynamics!lead!to!timeVdependent!changes!in!the!electronic!energy!gap!that!are!
resolvable!with!a!variety!of!nonlinear!techniques.!!
In!this!study,!the!origin!of!Stokes!shift!was!explored!along!a!lineage!from!
mKate!to!TagRFP675.!!SiteVdirected!mutagenesis!enables!targeting!of!specific!
sidechain!interactions!that!are!thought!to!facilitate!excited!state!stabilization!of!the!
chromophore.!!A!panel!of!mutants!was!devised!to!probe!the!hydrogen!bond!network!
surrounding!the!NVacylimine!end!of!the!chromophore.!!Crystal!structures!reveal!two!
unique!interactions,!waterVmediated!and!direct!hydrogen!bonds,!with!the!F62!
carbonyl!as!illustrated!in!Figure!4.1.!!The!Q106!sidechain!and!an!embedded!water!
molecule!compose!the!former,!while!the!latter!is!an!interaction!with!the!carboxylic!
acid!moiety!of!the!Q41!sidechain.!!Three!mutants!were!designed!to!investigate!the!
waterVmediated!hydrogen!bond:!Q106M,!F62A,!and!Q106M!F62A.!!It!is!predicted!
that!these!changes!will!effectively!disrupt!this!interaction!and!help!elucidate!its!role!
in!excited!state!stabilization.!!This!hydrogen!bond!motif!is!observed!in!other!farVred!
emitting!FPs!including!mPlum,!mNeptune,!and!mCardinal.!!Systematic!elimination!
and!addition!of!this!interaction!to!TagRFP675!(TagRFP675!Q41M)!and!mKate!
(mKate!M41Q)!respectively!demonstrate!its!role!in!extending!Stokes!shift.!!

106!
!
To!explore!hydrogen!bond!solvation!dynamics,!we!employed!SRTG!
spectroscopy!as!previously!applied!in!Chapter!III!to!investigate!excited!state!
solvation!stemming!from!the!extended!hydrogen!bond!network!of!the!native!RFPs!
and!strategic!point!mutants168.!!Such!information!will!help!precisely!characterize!
specific!chromophoreVsidechain!interactions!thought!to!influence!their!emission!
properties.!!MD!simulations!will!be!utilized!to!corroborate!the!experimental!results.!!
These!results!lend!greater!insight!into!the!molecular!mechanism!of!Stokes!shift!in!
farVred!emitting!RFPs!and!offer!the!opportunity!draw!a!comparison!with!our!
previous!findings!in!mPlum.!!Ultimately,!these!principles!might!possibly!help!guide!
the!engineering!of!more!robust!redVemitting!species.!
4.2(Experimental(Methods(
Time;Resolved#Spectroscopy#
Transient!grating!measurements!were!performed!as!previously!described!in!
Chapter!III!with!the!JILA!MONSTR!nonlinear!optical!platform106,!168.!!Upon!
irradiation!with!3!excitation!pulses!(20!fs,!~10!nJ/beam),!the!spatially!isolated!fourV
wave!mixing!signal!emitted!in!the!prescribed!ks=Vk1+k2+k3!phase!matching!
direction.!!Excitation!wavelength!was!chosen!to!maximize!contrast!between!ground!
state!bleach!and!stimulated!emission!components.!!The!sample!was!refreshed!with!a!
spinning!cell!to!mitigate!photobleaching!and!accumulation!of!laserVinduced!
photoproducts.!!Sample!concentration!and!path!length!were!again!carefully!tuned!to!
avoid!aggregation!and!reabsorption!of!the!nonlinear!signal.!!Concentrations!of!~50!
µM!and!a!path!length!of!0.5!mm!were!used!for!all!samples!(OD!0.15!at!peak!
absorption).!!SpectrallyVresolved!homodyne!detection!was!carried!out!with!a!liquid!

107!
!
N2Vcooled!CCD!camera.!!Data!was!collected!as!a!function!of!the!T!time!delay!(k1(and!
k2(temporally!overlapped)!from!a!range!of!0!to!1.3!ns,!spaced!logarithmically,!with!a!
background!spectrum!collected!at!V500!fs!delay!to!eliminate!contributions!from!
excitation!scatter!and!spontaneous!emission.!!The!baselined!spectral!data!was!fit!to!
a!sum!of!two!Gaussians!using!a!nonlinear!least!squares!fitting!algorithm.!!SE!peak!
center!position!and!amplitude!was!extracted!at!each!time!delay!and!fit!to!
multiexponential!decays!to!yield!solvation!information.!
Molecular#Dynamics#Simulations#
Time!series!trajectories!were!obtained!from!explicit!solvent,!allVatom!
simulations!using!the!NAMD!molecular!dynamics!package!with!the!CHARMM27!
force!field153V154.!!The!initial!XVray!crystallographic!structures!were!obtained!from!
the!Protein!Data!Bank!(mKate!pdb!code!3BXB,!TagRFP675!pdb!code!4KGF).!!The!
mutants!studied!do!not!have!PDB!structures,!so!we!used!CHARMM!to!make!the!
amino!acid!substitution!from!the!respective!parent!files.!!Force!field!parameters!for!
the!mature!chromophore!were!adopted!from!the!anionic!GFP!chromophore!
developed!by!Reuter!et#al.!and!from!CHARMM27!parameters!for!acylimine!
nitrogen155.!!Throughout!the!simulation,!the!deprotonated!anionic!form!of!the!
chromophore!in!the!ground!state!was!used.!!In!addition,!E215!was!protonated!using!
a!patch.!
The!VMD!package!was!used!to!setup!the!system!for!simulations156.!!The!
initial!structure!with!crystallographic!water!molecules!was!solvated!by!using!the!
solvate!plugin!in!VMD.!!For!all!mutants,!the!box!cutoff!was!set!to!10!Å.!!This!resulted!

108!
!
in!a!simulation!box!of!dimensions!66!x!72!x!77!Å3,!with!similar!dimensions!for!the!
other!mutants.!!The!solvated!system!was!electrically!neutralized!by!adding!five!Na+!
ions!randomly!in!the!bulk!water!using!the!VMD!autoionize!plugin.!!The!final!system!
contained!about!35000!atoms.!!All!water!molecules!overlapping!with!the!protein!
were!removed.!!The!particle!mesh!Ewald!method!was!used!to!treat!longVrange!
interactions!with!a!12!Å!nonbonded!cutoff157.!!Energy!minimization!was!performed!
using!the!conjugate!gradient!and!line!search!algorithm.!!The!system!was!then!heated!
for!90!ps!with!a!linear!gradient!of!20K/6ps!from!20!to!300!K.!!At!300!K,!the!system!
was!equilibrated!for!910!ps!with!a!2!fs!integration!time!step!in!the!NVT!(constant!
number,!volume,!and!temperature)!ensemble.!Langevin!dynamics!was!used!to!
maintain!the!temperature!at!300!K.!!The!production!run!was!50!ns!using!NVT!
dynamics!with!2!fs!time!steps.!!The!last!40!ns!of!the!production!run!was!used!for!
analysis!
Protein#Expression,#Mutagenesis,#and#Purification#
Fluorescent!proteins!were!expressed!with!the!pBAD!vector!containing!their!
corresponding!gene.!!Point!mutations!were!introduced!with!QuikChange!
mutagenesis!using!custom!designed!primers!(IDT).!!Expression!procedures!differed!
slightly!for!the!two!primary!lineages!studied.!!For!mKate!and!mutants,!plasmids!
were!transformed!into!Top10!E.#coli,!grown!to!OD600=0.6!at!37!°C,!followed!by!
induction!with!0.02%!arabinose!for!24!hours!at!30!°C.!Plasmids!of!TagRFP675!and!
mutants!were!transformed!into!the!LMG194!E.#coli!strain,!grown!to!OD600=0.6!at!
37!°C,!followed!by!induction!with!0.02%!arabinose!for!48!hours!at!18!°C.!!Samples!

109!
!
were!purified!via!6XVHis!tag/NiVNTA!chromatography!and!the!crude!specimens!
were!bufferVexchanged!with!15!mM!pH!8.0!TRIS!buffer,!100!mM!KCl.!!Emission!
spectra!of!each!mutant!were!detected!with!a!diode!array!spectrometer!and!its!peak!
value!helped!verify!the!purity!of!the!final!products.!
4.3(Results(
Steady#State#Spectroscopy#
The!linear!absorption!and!emission!data!are!tabulated!below.!The!absorption!
peaks!fall!between!585V600!nm!as!typically!observed!in!other!RFPs.!!The!emission!
values!also!show!systematic!trends.!!Addition!of!a!hydrogen!bond!at!the!NVacylimine!
carbonyl!in!mKate!M41Q!leads!to!an!18!nm!redVshifted!emission,!while!its!removal!
in!TagRFP675!Q41M!has!the!opposite!effect!yielding!a!32!nm!blueVshift!relative!to!
the!native!variant.!!Disruption!of!the!waterVmediated!hydrogen!bond!network!also!
leads!to!blueVshifted!emission!in!the!TagFP675!F62A,!Q106M,!and!F62A!Q106M!
mutants.!!!
Table(4.1.!!Steady!state!absorption!and!emission!data!for!all!species.!
Absorption&
Peak&(nm)&Emission&Peak&(nm)&

Stokes&Shift&41(cm )&

Quantum&Yield&

mKate&

588&

635&

1259&

0.30&

mKate&M41Q&

585&

653&

1780&

44&

TagRFP675&

600&

675&

1852&

0.08&

TagRFP675&F62A&

598&

658&

1525&

44&

TagRFP675&Q106M&

590&

648&

1517&

44&

Tag&RFP675&F62A&Q106M&

588&

642&

1430&

44&

TagRFP675&Q41M&

590&

643&

1397&

0.18&

Mutant&#110!
!
Time;Resolved#Spectroscopy#
The!experimental!results!for!all!species!are!presented!in!the!figures!below.!!
First,!contour!plots!illustrating!SRTG!spectra!and!fitting!results!are!presented!in!
Figures!4.2V4.4.!!Ground!State!Bleach!(GSB)!and!Stimulated!Emission!(SE)!
components!are!resolved!in!the!TagRFP675,!TagRFP675!F62A,!TagRFP675!Q106M,!
TagRFP675!F62A!Q106M,!and!mKate!M41Q!spectra.!!The!SE!component!is!not!
obvious!for!mKate!and!TagRFP675!Q41M.!!This!is!presumed!to!be!an!effect!of!a!blueV
shifted!Excited!State!Absorption!(ESA)!band!leading!to!greater!overlap!with!the!SE!
component.!!It!is!interesting!to!note!that!this!effect!occurs!only!in!variants!lacking!a!
hydrogen!bond!interaction!between!the!41!sidechain!and!the!NVacylimine!carbonyl!
and!suggests!it!has!a!role!in!tuning!RFP!electronic!structure!beyond!the!first!singlet!
excited!state.!!!
Numerical!fitting!results!and!error!analysis!are!tabulated!below!in!Tables!4.2!
and!4.3.!!In!general,!these!results!can!be!categorized!as!follows.!!No!solvation!is!
evident!for!mKate!and!TagRFP675!Q41M!judging!by!the!stationary!GSB!component!
and!lack!of!spectral!evolution!on!the!low!energy!side!of!the!SRTG!data.!!All!other!
species!show!a!consistent!biexponential!decay!behavior.!!The!early!component!is!
slightly!faster!for!mKate!M41Q,!TagRFP675!F62A,!and!TagRFP675!Q106M!(4.5V7.1!
ps)!compared!to!TagRFP675!and!TagRFP675!F62A!Q106M!(17.5!and!24!ps).!!The!
slower!component!can!be!divided!into!three!groups:!537!ps!(TagRFP675!F62A!
Q106M),!355V396!ps!(TagRFP675,!mKate!M41Q),!and!159V216!ps!(TagRFP675!F62A,!
TagRFP675!Q106M).!!Ultrafast!solvation!appears!evident!in!all!mutants,!but!its!
measurement!is!hindered!by!pulse!overlap!effects!apparent!within!the!first!~200!fs.!!

111!
!
The!shift!magnitudes!were!proportionally!larger!for!the!slower!timescale!in!all!cases!
except!for!TagRFP675!F62A!Q106M.!!This!mutant!and!TagRFP675!Q106M!showed!
the!smallest!combined!shifts!of!35!cmV1.!!The!remaining!mutants!exhibited!combined!
shifts!of!75V110!cmV1.!!!!!!

a

650

600

550
0

200

400
600
Delay (ps)

800

c

700

600

200

400
600
Delay (ps)

800

600

550
0

650

550
0

650

1000

Wavelength (nm)Wavelength (nm)

700

1000

b

700

Wavelength (nm)Wavelength (nm)

700

200

400
600
Delay (ps)

800

1000

d

650

600

550
0

200

400
600
Delay (ps)

800

1000

!
Figure(4.2.!!SRTG!spectra!of!(a)!TagRFP675,!(b)!TagRFP675!Q41M,!(c)!mKate,!and!
(d)!mKate!M41Q.!
!
!

112!
!

15180

15540

b
SE Peak Frequency (cm-1)

15160

SE Peak Frequency (cm-1)

a

Data
Fit

15140
15120
15100
15080
15060
15040
15020

0

100

200
300
Delay (ps)

400

500

15520
15500
15480
15460
15440
15420
15400

0

200

400
Delay (ps)600!

Figure(4.3.!!SE!band!peak!positions!extracted!from!double!Gaussian!fitting!of!SRTG!
spectra!and!multiexponential!decay!fits!for!(a)!TagRFP675!and!(b)!mKate!M41Q.!
a

600

550
0

200

400
600
Delay (ps)

800

650

600

550
0

1000

15800

200

400
600
Delay (ps)

650

600

550
0

1000

200

400
600
Delay (ps)

800

1000

15780

e

15750

15700

15650

15600

15760
15740
15720
15700
15680

f

15820
SE Peak Frequency (cm-1)

SE Peak Frequency (cm-1)

d
SE Peak Frequency (cm-1)

800

c

700

Wavelength (nm)

650

b

700

Wavelength (nm)Wavelength (nm)

700

15800
15780
15760
15740
15720

15550

0

200

400
Delay (ps)

600

15660

0

200

400
Delay (ps)

600

0

200

400
Delay (ps)600

Figure(4.4.!!SRTG!spectra!and!fitted!SE!band!positions!for!various!time!delays!of!(a)!
TagRFP675!F62A,!(b)!TagRFP675!Q106M,!(c)!TagRFP675!Q106M!F62A.!
!
!
!
!

!

113!
!
Table(4.2.!!Fitting!parameters!assuming!multiexponential!shift!of!SE!band.!
Amplitude&
41(cm )&

τ1&(ps)&Amplitude&
41(cm )&

τ2&(ps)&mKate&

44&

44&

44&

44&

mKate&M41Q&

30&

5&

52&

355&

TagRFP675&

33&

17&

44&

397&

TagRFP675&Q41M&

44&

44&

44&

44&

TagRFP675&F62A&

41&

7&

68&

159&

TagRFP675&Q106M&

12&

4&

24&

219&

Tag&RFP675&F62A&Q106M&

18&

24&

17&

537&

Mutant&(Table(4.3.!!Fitting!error!analysis!for!multiexponential!fits!to!SE!band!shifts.!!The!
given!values!represent!95%!confidence!intervals!for!the!parameters!of!the!fit!
equation:! f (t ) = a ⋅ e−bt + c ⋅ e−dt .!!Amplitudes!are!expressed!in!wavenumbers!(cmV1)!
and!time!constants!are!reported!as!eV1!lifetimes!in!picoseconds.!!!!
41

Mutant&a&(cm )&

41

b&(ps)&

c&(cm )&

d&(ps)&

mKate&

44&

44&

44&

44&

44&

44&

44&

44&

mKate&M41Q&

27&

33&

4&

7&

50&

55&

317&

404&

TagRFP675&

27&

39&

12&

31&

38&

50&

309&

552&

TagRFP675&&
Q41M&

44&

44&

44&

44&

44&

44&

44&

44&

TagRFP675&&
F62A&

36&

47&

5&

10&

63&

73&

138&

186&

TagRFP675&&
Q106M&

8&

15&

3&

15&

22&

27&

177&

286&

Tag&RFP675&&
F62A&Q106M&

14&

22&

16&

46&

13&

21&

363&

1033&

!
(
(

114!
!
Table(4.4.!!Amplitude!decay!fits!of!SE!band!extracted!from!SRTG!spectra!for!
variants!demonstrating!an!excited!state!response.!
Mutant&Amplitude&

τ1&(ps)&Amplitude&

τ2&(ps)&mKate&

44&

44&

44&

44&

mKate&M41Q&

0.15!

2598!

0.85!

349!

TagRFP675&

0.17!

467!

0.83!

159!

TagRFP675&Q41M&

44&

44&

44&

44&

TagRFP675&F62A&

0.77!

269!

0.23&

39!

TagRFP675&Q106M&

0.35!

700!

0.65!

214!

Tag&RFP675&F62A&Q106M&

0.14!

407!

0.86!

15!

!
The!SE!band!amplitude!for!each!mutant!(Table!4.4),!integrated!over!all!
wavelengths,!versus!time!delay!were!determined!from!fits!to!a!multiexponential!
decay.!!This!value!should!reflect!the!fluorescence!lifetime!previously!reported!as!
900!ps!for!TagRFP67550.!!The!SRTG!SE!lifetimes!appear!2x!shorter!due!to!the!effect!
of!homodyne!detection.!!Considering!this!adjustment,!the!results!here!are!in!
qualitative!agreement!with!previous!findings!with!a!few!exceptions.!!A!lifetime!
component!of!~1!ns!was!observed!in!most!species.!!Their!multiexponential!
character!with!both!faster!and!slower!timescales!alludes!to!complex!photophysical!
behavior!predicted!for!RFPs146.!!Variation!among!the!point!mutants!is!observed!and!
is!not!surprising!given!the!effect!of!disrupting!local!chromophoreVsidechain!
interactions!on!other!spectral!properties.!!
MD#Simulations#
The!timescales!extracted!from!the!SE!SRTG!response!report!directly!on!the!
excited!state!nuclear!reorganization!thought!to!contribute!to!the!extended!Stokes!

115!
!
shift!of!farVred!emitting!RFPs,!including!TagRFP675.!!We!utilized!MD!simulations!to!
connect!the!experimental!results!to!specific!chromophoreVsidechain!interactions.!!
While!a!fully!quantum!mechanical!treatment!is!required!to!model!an!excited!
electronic!state,!classical!MD!simulations!are!considered!a!valid!method!to!capture!
the!timescales!of!molecular!motions!contributing!to!excited!state!solvation!within!
the!linear!response!approximation162.!!!
MD!trajectories!of!each!mutant!were!carried!out!to!examine!chromophore!
flexibility!and!the!possibility!of!interconversion!between!hydrogen!bond!states!as!
previously!observed!in!mPlum.!!These!modeling!results!represent!only!a!
preliminary!investigation!of!the!TagRFP675!system.!!Further!analysis!including!
simulations!on!the!remaining!mutants!is!ongoing!and!will!be!included!in!a!future!
manuscript.!!We!first!examined!the!dynamics!of!the!interaction!at!positions!62!and!
41!in!TagRFP675!and!mKate!M41Q.!!In!Figure!4.5,!the!time!trajectories!of!the!
proximity,!expressed!as!Δr,!between!the!participating!atom!in!the!Q41!sidechain!and!
the!NVacylimine!oxygen!of!residue!F62.!!It!is!apparent!that!both!variants!occupy!only!
single!conformation!throughout!the!50!ns!trajectory,!which!is!identified!as!direct!
hydrogen!bond!through!analysis!of!the!output!coordinates.!!This!is!in!contrast!to!the!
behavior!observed!in!mPlum.!!

116!
!

7

7
b

6

6

5

5
r (Å)r (Å)

a

4

4

3

3

2

2

1

0

10

20
30
Time (ns)

40

50

1

0

10

20
30
Time (ns)

40

50

!

Figure(4.5.!!Time!trajectories!of!the!Q41Vacylimine!carbonyl!distance!(Δr)!for!(a)!
TagRFP675!and!(b)!mKate!M41Q.!!Δr!refers!to!the!distance!between!the!sidechain!of!
Q41!and!the!NVacylimine!oxygen!of!F62.!
The!hydrogen!bond!behavior!between!S28!and!Q41!was!also!assessed.!!This!
interaction!was!identified!in!the!crystal!structure!and!was!speculated!to!play!a!role!
in!stabilizing!the!interaction!between!Q41!and!F62.!!The!MD!trajectory!presented!in!
Figure!4.6!illustrates!unique!results!for!the!two!species.!!TagRFP675!exhibits!
intermittent!conversion!between!two!discrete!proximities,!while!mKate!M41Q!
primarily!occupies!the!closest!substate.!!These!results!are!also!depicted!as!
probability!histograms!in!Figure!4.7.!!

117!
!

7

7
b

6

6

5

5

r (Å)r (Å)

a

4

4

3

3

2

2

1

0

10

20
30
Time (ns)

40

1

50

0

10

20
30
Time (ns)

40

50

(

Figure(4.6.((Time!trajectories!of!the!S28VQ41!distance!(Δr)!for!(a)!TagRFP675!and!
(b)!mKate!M41Q.!!Δr!is!defined!as!the!distance!between!the!sidechains!of!S28!and!
Q41.!
0.09

0.15
TagRFP675
mKate M41Q

62-41

0.08

41-28

0.07
0.1

Probability

Probability

0.06
0.05
0.04
0.03

0.05

0.02
0.01
0

0

2

4
r (Å)

6

8

0

1

2

3

4
r (Å)

5

6

7(

Figure(4.7.!!Histograms!of!the!probability!for!the!Q41VF62!and!Q41VS28!separations,!
Δr,!to!occur!during!the!MD!simulations.!!!
The!two!substates!for!the!TagRFP675!41V28!proximity!were!identified!as!direct!and!
waterVmediated!hydrogen!bonds!through!coordinate!analysis.!!The!waterVmediated!
bond!forms!following!intermittent!rotation!of!the!S28!sidechain!and!simultaneous!
migration!of!a!water!molecule.!!The!distance!distributions!were!broader!for!
TagRFP675!indicating!greater!sidechain!flexibility,!which!may!facilitate!this!

118!
!
structural!rearrangement.!!The!prevalence!of!participating!hydrogen!bonds!to!the!
chromophore!for!all!mutants!is!tabulated!below.!!Percentages!are!mostly!consistent!
across!all!mutants!aside!from!the!41V62!interaction!reaffirming!its!importance!for!
driving!excited!state!stabilization.!!!!!!
Table(4.5.((HydrogenVbonding!to!the!chromophore!from!neighboring!sidechains!
presented!in!terms!of!time!fraction!as!calculated!from!MD!simulations.!!For!each!
mutant,!bonds!with!sidechains!Q41,!Q106,!W90,!and!S28!are!reported.!!For!all!
entries,!3.5!Å!distance!and!30°!angle!cutVoff!values!were!used!for!defining!a!
hydrogen!bond.!
41462&(%)&

1064H2O&(%)&

28441&(%)&

H2O4CHR&(%)&

W904CHR&(%)&&
TagRFP675&

49&

74&

81&

34&

57&

mKate&

44&

79&

44&

32&

66&

mKate&M41Q&

63&

81&

83&

32&

70&

#
4.4(Discussion(Specific!hydrogenVbonding!interactions!identified!in!crystal!structures!have!
been!implicated!as!the!origin!of!the!extended!Stokes!shift!of!TagRFP675.!!Two!
hydrogen!bonds!were!identified!at!the!NVacylimine!carbonyl!end!of!the!
chromophore!as!illustrated!in!Figure!4.1.!!The!timeVresolved!spectroscopy!results!
help!shed!light!on!the!mechanism!of!excited!state!stabilization!and!relative!
importance!of!these!interactions.!!The!SRTG!data!indicate!picosecond!solvation!
previously!attributed!to!similar!hydrogen!bond!dynamics!in!mPlum168.!!However,!
distinct!behavior!is!observed!among!the!different!mutants!studied.!!Variants!lacking!
a!direct!hydrogen!bond!with!the!Q41!sidechain!(mKate,!TagRFP675!Q41M)!show!no!
clear!solvation!on!the!experimental!timescale.!!Addition!of!this!interaction!to!mKate!
with!a!single!M41Q!mutation!leads!to!a!dramatically!larger!Stokes!shift!on!par!with!

119!
!
TagRFP675!and!nearly!equivalent!solvation!dynamics.!!Disruption!of!the!waterV
mediated!hydrogen!bond!between!Q106!and!the!NVacylimine!was!attempted!with!
the!TagRFP675!F62A,!Q106M,!and!F62A!Q106M!mutants.!!A!~300!cmV1!smaller!
Stokes!shift!is!observed!for!each!of!these!species!that!reflects!the!removal!of!this!
bond.!!Oddly,!this!perturbation!seems!to!have!only!a!small!effect!on!overall!solvation!
dynamics!where!a!modest!increase!in!the!slow!relaxation!was!observed!for!F62A!
and!Q106M.!!We!attribute!this!difference!to!a!change!in!flexibility!of!the!NVacylimine!
carbonyl!given!a!reduction!in!steric!hindrance!of!the!phenylalanine!ring!in!F62A!and!
loss!of!the!waterVmediated!hydrogen!bond!in!Q106M.!!These!results!suggest!that!the!
direct!hydrogen!bond!at!the!NVacylimine!carbonyl!has!the!highest!impact!on!
extending!Stokes!shift!in!TagRFP675.!!!
The!hydrogen!bond!behavior!of!TagRFP675!is!strikingly!different!than!
observed!for!mPlum,!where!interconversion!between!two!unique!conformations!
was!predicted!at!the!NVacylimine!position,!which!appeared!to!correlate!with!the!
magnitude!of!the!Stokes!shift!among!a!panel!of!point!mutants168.!!Investigation!of!
this!dynamic!component!was!similarly!carried!out!in!this!study.!!The!MD!modeling!
results!shown!in!Figure!4.5!suggest!that!the!hydrogen!bond!with!Q41!does!not!
undergo!conformational!interconversion!on!the!timescales!observed!in!mPlum.!!An!
identical!analysis!was!conducted!for!the!waterVmediated!hydrogen!bond!with!Q106,!
which!demonstrated!slow!~ns!sidechain!rotation!in!TagRFP675.!!A!similar!
conformational!change!was!predicted!for!mKate!and!mKate!M41Q,!however,!they!
exhibited!fast!(<ps)!recovery!to!the!original!hydrogenVbonded!state.!!It!is!doubtful!
that!these!dynamics!alone!explain!the!redVshifted!emission!of!TagRFP675!relative!to!

120!
!
the!parent!mKate!since!its!disruption!accounts!for!only!a!small!portion!of!the!overall!
reorganization!energy!(~300!cmV1)!and!its!timescale!is!slower!than!the!excited!state!
lifetime!(~900!ps).!!Flexibility!at!this!position!is!more!likely!to!account!for!its!
reduced!quantum!yield!relative!to!the!parent!species!either!through!internal!
conversion!or!chromophore!cisVtrans!isomerization!evident!in!crystal!structures.!!
The!conformational!interconversion!predicted!at!the!Q41VS28!position!is!an!
interesting!result.!!It!is!possible!that!this!interaction!indirectly!modulates!the!
hydrogen!bond!strength!of!Q41VF62!and!thus,!chromophore!flexibility!in!a!manner!
similar!to!mPlum.!!Alternatively,!this!behavior!may!merely!reflect!a!more!flexible!
local!environment!leading!to!more!robust!solvation!of!the!excited!chromophore.!!(Our!results!further!shed!light!on!the!role!of!the!NVacylimine!in!tuning!
electronic!properties!of!DsRedVlike!RFPs.!!Hydrogen!bond!analysis!from!MD!
simulations!(Table!4.5)!reveals!little!variation!of!interactions!at!other!sites!on!the!
chromophore!among!the!mutants!studied.!!Its!significant!impact!is!surprising!given!
its!location!at!the!end!of!the!conjugated!ring!system.!!A!few!possibilities!exist!to!
explain!this!behavior.!!First,!the!direct!hydrogen!bond!with!neighboring!sidechains!
may!act!as!an!anchor!to!hold!the!carbonyl!in!the!plane!of!conjugation!and!facilitate!
stabilization!of!the!excited!state.!!Alternatively,!this!anchor!may!act!to!restrain!the!
entire!imidazolinone!ring!and!help!maintain!coplanarity!across!the!entire!
conjugated!system.!!This!model!is!counterintuitive!since!a!substantial!decrease!in!
quantum!yield!occurs!in!species!having!this!interaction.!!A!second!consideration!is!
flexibility!at!this!end!of!the!chromophore.!!In!principle,!solvation!is!driven!by!
environmental!flexibility!capable!of!conforming!to!a!new!excited!state!charge!

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!
distribution.!!The!extent!of!this!process,!however,!might!be!restricted!by!nuclear!
degrees!of!freedom!of!the!donor!and!acceptor!groups.!!An!example!of!this!is!
observed!with!the!F62A!mutant,!which!shows!a!~300!cmV1!loss!of!Stokes!shift!
compared!to!TagRFP675,!however,!is!predicted!to!retain!both!direct!and!waterV
mediated!hydrogen!bonds!at!the!NVacylimine!position.!!Moreover,!this!mutation!
perturbs!only!the!excited!state!(emission!wavelength)!and!its!accelerated!solvation!
compared!to!the!parent!is!consistent!with!a!change!in!flexibility!at!this!position!
possibility!due!to!lessor!steric!hindrance!with!the!loss!of!the!bulky!phenylalanine!
sidechain.!
These!results!may!also!be!interpreted!in!the!context!of!designing!more!
robust!RFPs.!!More!specifically,!developing!desirable!variants!with!both!red!
emission!wavelength!and!high!fluorescence!quantum!yield.!!This!goal!might!be!
achieved!in!a!few!ways.!!First,!Stokes!shift!can!be!increased!through!solvation!via!
tertiary!interactions!(e.g.!hydrogen!bonds!or!πVπ!stacking),!however,!a!pliable!
chromophore!environment!is!most!typically!accompanied!with!a!sharp!drop!in!
quantum!yield!due!to!increased!nonradiative!deactivation.!!It!is!unknown!whether!
the!hydrogen!bond!interconversion!observed!in!mPlum!might!provide!an!additional!
route!for!extending!redVemission!in!TagRFP675.!!This!effect!might!be!modulated!by!
secondary!hydrogen!bonds!that!regulate!the!range!of!motion!of!the!Q41!sidechain!
itself.!!In!TagRFP675,!S28!forms!a!hydrogen!bond!with!Q41!while!the!E16!sidechain!
in!mPlum!is!unrestricted!by!a!second!interaction.!!The!contrasting!behavior!between!
TagRFP675!and!mKate!M41Q!at!this!position!identified!in!the!MD!simulations!
suggests!it!might!play!a!role!tuning!its!photophysical!properties.!!Second,!red!

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!
emission!may!be!achieved,!even!with!a!moderate!Stokes!shift,!with!a!simultaneous!
shift!in!absorption!and!emission!wavelength.!!This!occurs!in!closely!related!variants!
mNeptune!and!mCardinal,!which!demonstrate!emission!around!650!nm!with!~3!
fold!higher!quantum!yield!compared!to!TagRFP675.!!Finally,!a!third!possible!
strategy!for!RFP!optimization!involves!a!combination!of!the!previous!models.!!
Identifying!structural!channels!of!nonradiative!deactivation!that!are!particularly!
sensitive!to!quantum!yield!will!be!a!critical!challenge!in!this!effort.!!Minimizing!this!
effect!while!exploiting!environmental!interactions!that!drive!excited!state!
stabilization!will!be!essential!to!achieving!this!goal.!!Nonlinear!spectroscopic!
techniques!capable!of!quantifying!the!dynamics!of!the!chromophore!and!its!local!
environment!together!with!theoretical!modeling!and!targeted!mutagenesis!are!
uniquely!suited!for!guided!exploration!of!new!RFP!species.!
4.5(Conclusions(
The!excited!state!relaxation!of!the!farVred!emitting!FP!TagRFP675!and!a!
panel!of!strategic!point!mutants!was!quantified!with!SRTG!spectroscopy.!!We!found!
that!biexponential!solvation!occurred!on!~10s!and!100s!of!ps!timescales.!!This!
behavior!was!attributed!to!hydrogen!bond!interactions!between!the!NVacylimine!
carbonyl!of!the!chromophore!and!the!neighboring!sidechains!Q106!and!Q41!on!the!
basis!of!MD!simulations.!!In!TagRFP675!and!mKate!M41Q,!a!direct!hydrogen!bond!
was!identified!between!F62!and!Q41.!!While!removal!of!this!interaction!in!
TagRFP675!Q41M!and!mKate!coincides!with!a!sharply!blueVshifted!emission!
wavelength!and!loss!of!transient!spectral!behavior!and!implicates!its!role!in!
extending!Stokes!shift,!the!exact!mechanism!of!this!is!unclear.!!Preliminary!MD!

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analysis!indicates!TagRFP675!and!mKate!M41Q!do!not!undergo!hydrogen!bond!
interconversion!at!the!F62VQ41!position!in!contrast!to!previous!observations!of!
mPlum.!!However,!the!supporting!S28VQ41!hydrogen!bond!of!TagRFP675!is!
predicted!to!undergo!similar!conformational!change.!!This!behavior!suggests!
interactions!at!the!acylimine!end!of!the!chromophore!might!act!to!modulate!its!
flexibility!and!are!essential!for!achieving!redVshifted!emission.!!Additional!modeling!
of!these!results!will!focus!on!better!quantifying!chromophore!flexibility!among!the!
remaining!variants.!!These!results!further!understanding!of!the!mechanism!of!red!
emission!in!RFPs!and!dynamics!of!buried!probes!in!protein!systems.!
!
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!
!
!
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!
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!

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!
Chapter(V(Excited(State(Electronic(Landscape(of(mPlum(Revealed(by(TwoZDimensional(
Double(Quantum(Coherence(Spectroscopy(
As!Appeared!in!the!Journal!of!Physical!Chemistry!B,!2015,!Vol.!119,!3414V3422!

!
5.1(Introduction(!

Given!the!combination!of!their!complex!photophysics!and!straightforward!

physical!structure,!FPs!serve!as!an!ideal!model!system!for!investigating!the!
electronic!structure!of!buried!optical!probes.!!GFP!has!garnered!most!attention!to!
date.!!It!exhibits!a!high!fluorescence!quantum!yield!and!reversible!photobleaching!
thought!due!to!excited!state!proton!transfer!involving!the!pVhydroxybenzylidene!
moiety169.!!TimeVresolved!spectroscopy!has!been!instrumental!in!uncovering!
intermediates!within!this!photocycle!having!lifetimes!ranging!from!femtoseconds!to!
milliseconds8,!170.!!The!study!of!red!fluorescent!protein!(RFP)!electronic!structure,!
however,!has!remained!largely!unexplored.!!Several!plausible!hypotheses!exist!to!
explain!the!photobleaching!phenomena!in!RFPs!including!conformational!
fluctuations!about!the!methylidene!bridge,!triplet!state!formation,!and!increased!
oxygen!permeability!stemming!from!monomerization!of!the!progenitor!DsRed16,!171.!!
Much!effort!has!focused!on!understanding!interactions!between!the!surrounding!
environment!and!positions!of!the!ground!and!first!excited!state!and!their!
association!with!emission!efficiency45.!!However,!a!detailed!understanding!of!the!
electronic!structure!underlying!these!photophysics!and!their!relationship!with!the!
nature!of!the!local!protein!environment!is!largely!unexplored.!!!
!

A!broader!knowledge!of!FP!excited!electronic!structure!also!has!practical!

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!
implications!for!advancing!imaging!applications.!!For!example,!multiphoton!
excitation!techniques!rely!heavily!on!the!position!and!strength!of!electronic!
excitation!into!single!or!doubly!excited!states172.!!However,!predicting!the!position!
and!strength!of!these!transitions!is!not!straightforward!given!the!diverse!
mechanisms!of!environmental!coupling!among!a!large!and!increasing!set!of!known!
FPs148,!173V174.!!Two!photon!absorption!(2PA)!studies!have!shed!light!on!these!
transitions!in!several!FPs!and!much!debate!exists!over!the!peak!structure!of!their!
spectra175V176.!!Features!often!attributed!to!oneVphoton!transitions!are!slightly!blueV
shifted!from!the!maxima!of!the!linear!absorption!spectra.!!Drobizhev!et#al.!explained!
this!shift!in!terms!of!vibronic!coupling177.!!Moreover,!2PA!is!also!observed!at!
energies!higher!than!the!expected!S0VS1!region!in!some!variants.!!These!were!
assigned!as!transitions!to!higher!lying!electronic!states,!S0VSn,!and!their!unusually!
large!cross!sections!were!attributed!to!nearVresonance!enhancement!known!to!
occur!in!large!aromatic!systems175.!!We!recently!reported!on!the!timeVresolved!
solvation!dynamics!of!a!series!of!mPlum!mutants,!where!an!ESA!contribution!
observed!on!the!low!energy!side!of!GSB!led!to!a!reduction!in!the!magnitude!of!redV
shift!by!the!stimulated!emission!contribution168.!!This!observation,!along!with!
another!transient!absorption!study!confirms!the!existence!of!nearby!Sn!states,!but!
did!not!aim!to!precisely!determine!their!transition!energies!or!intensities178.!!!
!

In!recent!years,!sophisticated!coherent!multidimensional!spectroscopic!

techniques!have!been!developed!to!effectively!spread!an!absorption!spectrum!over!
two!frequency!axes!whose!time!dependence!can!be!monitored!with!femtosecond!
precision110.!!This!information!has!proven!invaluable!for!deciphering!complex!

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!
electronic!structure!in!congested!spectral!regions!and!energy!transfer!events!within!
multichromophoric!systems!such!as!photosynthetic!light!harvesting!complexes69.!!
The!utility!of!analogous!NMR!and!vibrational!spectroscopic!methods!has!been!
routinely!demonstrated!for!many!years,!but!only!recently!with!electronic!transitions!
at!optical!frequencies!given!the!challenges!associated!with!femtosecond!pulse!
generation!and!subcycle!phase!control110.!!
!

The!most!frequently!employed!2D!electronic!spectroscopy!method!examines!

the!correlation!between!absorption!and!emission!frequencies!centered!about!the!
energy!of!the!fundamental!electronic!transition!(i.e.!from!ground!the!to!first!excited!
state).!!A!closelyVrelated!third!order!optical!response!probes!double!quantum!
pathways!that!lead!to!oscillations!near!twice!the!incident!laser!frequency179.!!For!a!
typical!molecular!system,!this!roughly!overlaps!with!a!twoVelectron!state!resulting!
in!coherence!of!a!double!quantum!resonance.!!Exposure!to!a!subsequent!third!pulse!
yields!signals!projected!from!its!constituent!single!quantum!resonances!leading!to!a!
spectrum!that!correlates!single!and!double!quantum!transitions.!!This!technique!has!
been!used!in!the!mid!infrared!to!decipher!protein!structures!in!biological!systems,!
manyVbody!effects!in!semiconductor!quantum!wells,!and!electron!correlation!effects!
in!organic!dyes180V185.!!For!a!three!level!system!(3LS),!in!a!ladder!configuration,!the!
double!quantum!spectrum!exhibits!two!peaks!of!opposite!sign,!when!projected!on!
the!emission!axis,!that!represent!the!0V1!and!1V2!energy!gaps.!!The!inclusion!of!
additional!states!and!vibrational!modes!leads!to!additional!responses!and!
complicate!the!peak!shapes186.!

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!
!

In!this!study,!we!find!that!2D2Q!spectra!of!mPlum!are!distinctly!different!from!

those!of!cresyl!violet!in!methanol,!previously!well!described!as!a!3LS.!!A!
combination!of!excitation!dependent!2D2Q,!SRTA!spectra,!and!model!calculations!
suggest!mPlum!is!most!consistent!with!a!1V1V2!electronic!level!structure,!with!two!
levels!in!the!doubly!excited!manifold!with!opposite!anharmonicity.!!These!results!
are!discussed!in!the!context!of!known!RFP!excitedVstate!properties.!!(5.2(Experimental(Methods(
Multidimensional#Spectroscopy#!

Double!Quantum!TwoVDimensional!Fourier!Transform!Spectroscopy!(2D2Q)!

was!carried!out!in!the!BOXCARs!geometry!with!excitation!and!local!oscillator!beams!
arranged!on!the!corners!of!a!1Vinch!square.!!Upon!irradiation!with!3!excitation!
pulses!(~2!nJ/beam,!20!kHz),!generated!from!a!noncollinear!optical!parametric!
amplifier!(20!fs,!WyvernV20!KM!Labs),!the!spatially!isolated!four!wave!mixing!signal!
is!emitted!in!the!prescribed!ksig=k1+k2Vk3!phase!matching!direction.!!The!sample!
was!contained!between!two!fused!silica!windows!(1Vinch!diameter,!1!mm!thick)!
separated!by!a!250!µm!Teflon!spacer!and!was!refreshed!with!a!spinning!cell!rotating!
at!3000!rpm!to!mitigate!photobleaching!and!accumulation!of!laserVinduced!
photoproducts.!!SpectrallyVresolved!detection!was!carried!out!with!a!liquid!N2V
cooled!CCD!camera.!!This!dimension!represents!the!emission!frequency,!Ωemis.!!Data!
was!collected!as!a!function!of!the!t2!time!delay!in!0.89!fs!increments!and!subjected!
to!Fourier!analysis!yielding!Ωexc!frequency!information.!!

128!
!

Ωexc

Ωemis
LO

t1=0

t2

t3
Time

!

Figure(5.1.((Excitation!pulse!sequence!used!to!generate!the!third!order!nonlinear!
optical!signal.(!

Phase!stability!of!the!temporal!delays!between!pulses!within!the!excitation!

sequence!(Figure!5.1)!is!required!to!execute!heterodyne!detection!and!temporal!
sampling!with!interferometric!precision.!!This!stability!is!exceedingly!important!in!
the!optical!regime!where!environmental!drifts!may!cause!appreciable!phase!
fluctuations!within!the!experimental!time!interval.!!To!minimize!this!effect,!we!
employed!an!active!phase!stabilization!system!where!pathlengths!are!controlled!in#situ!by!piezoVdriven!mirrors!inside!the!JILAVMONSTR!nonlinear!spectroscopic!
platform106.!!Typical!phase!stabilities!exceeded!λ/200.!!!
The!raw!spectral!data!for!all!time!delays!was!compiled!into!a!single!matrix!
for!data!processing!and!normalized!with!the!square!root!of!the!local!oscillator!
intensity.!!Detected!wavelength!data!was!resampled!to!the!frequency!domain.!!Next,!
spectral!filtering!along!the!emission!frequency!dimension!was!conducted!using!
Fourier!analysis!to!remove!extraneous!signal!components.!!Finally,!Fourier!
transformation!along!the!t2!dimension!was!carried!out!to!extract!excitation!
frequency!information.!!The!excitation!frequency!vector!was!calculated!in!
accordance!with!the!Nyquist!theorem!assuming!a!temporal!sampling!interval!of!0.89!
fs.!!!!!

129!
!
Sample#Preparation#!

Samples!of!mPlum!were!purified!from!a!bacterial!expression!system!based!on!

the!pBAD!vector.!!Expression!plasmids!were!transformed!into!Top10!E.!coli!followed!
by!induction!with!0.02%!arabinose!for!24!hours.!!Purification!via!6XVHis!tag/NiVNTA!
chromatography!was!carried!out!and!samples!were!subsequently!bufferVexchanged!
with!15!mM!pH!8.0!TRIS!buffer,!100!mM!KCl.!!Linear!absorption!and!emission!
spectra!verified!the!purity!of!the!final!product.!!Cresyl!violet!was!purchased!from!
SigmaVAldrich!and!used!as!received.!!Samples!were!dissolved!in!spectroscopic!grade!
methanol.!!Sample!concentrations!were!carefully!selected!to!avoid!aggregation!and!
reabsorption!of!the!nonlinear!signal!(0.15!OD,!~50!µM).!
5.3(Results!
Steady#State#Spectroscopy#!

Steady!state!absorption!and!emission!spectra!are!shown!in!Figure!5.2.!!!

Normalized Amplitude (a.u.)

1.2

Emission
Absorption

1
0.8
0.6
0.4
0.2
0
1.4

1.5

1.6

1.7

1

Frequency (cm )

1.8

1.9
4
x 10

!

Figure(5.2.((Steady!state!absorption!and!emission!spectra!of!mPlum.((!

130!
!
Peak!positions!of!16978!and!15408!cmV1!respectively!are!consistent!with!those!
previously!reported44.!!mPlum!SRTA!spectra!collected!with!center!excitation!
wavelengths!of!16447!and!17241!cmV1!are!presented!in!Figure!5.3.!!Excitation!
dependence!reveals!strong!positive!and!negative!features!reflecting!Ground!State!
Bleach!(GSB)!and!Excited!State!Absorption!(ESA)!contributions.!!The!GSB!position!
closely!matches!the!linear!absorption!peak!as!expected,!however!it!is!slightly!
obscured!by!an!overlapping!ESA!feature.!!Interestingly,!the!relative!position!of!ESA!
to!GSB!differs!with!excitation,!falling!at!both!higher!and!lower!energies.!!These!
spectra!suggest!the!existence!of!two!ESA!bands!although!we!cannot!immediately!
discount!the!possibility!of!a!single!broad!overlapping!ESA!using!this!oneVdimensional!technique.!!!

1.5

1.5

SRTA
EXC

a

1

T/T (a.u.)

T/T (a.u.)

1

SRTA
EXC

b

0.5

0

1.5

1.6

1.7

1.8

Frequency (cm )

1.9

2
4

x 10

0.5

0

1.5

1.6

1.7

1.8

Frequency (cm )

1.9

2
4

x 10

!

Figure(5.3.((SRTA!spectra!of!mPlum,!with!center!excitation!wavelength!of!!(a)!17241!
and!(b)!16447!cmV1.!!Excited!state!absorption!components!(ΔT/T<0)!are!evident!on!
either!side!of!the!GSB!band.!
Multidimensional#Spectroscopy#!

2D2Q!data!for!cresyl!violet!in!methanol!and!mPlum!are!reported!in!Figures!4.4!

131!
!
and!5.5.!!For!cresyl!violet,!the!absolute!value!spectrum!exhibits!a!single!
inhomogeneously!broadened!peak!with!intensity!spanning!across!the!diagonal!in!
close!agreement!with!that!previously!reported183.!!

3.6 10a4

3.6 10b
3.5

exc (cm-1)

exc (cm-1)

3.5
3.4
3.3
3.2
3.1
1.55

4

3.4
3.3
3.2

1.6

1.65
1.7
emis (cm-1)

1.75
1.8
10 4

3.1
1.55

1.6

1.65
1.7
emis (cm-1)

1.75
1.8
10 4

!

Figure(5.4.!!(a)!Absolute!value!and!(b)!real!2D2Q!spectra!of!cresyl!violet!in!
methanol.!!
The!real!component!is!consistent!with!the!expected!3LS!peak!arrangement!with!a!
single!positive!and!negative!feature!above!and!below!the!diagonal,!respectively,!
representing!the!two!optical!responses!respectively!with!positive!anharmonicity.!!
The!mPlum!spectrum!is!strikingly!different!from!cresyl!violet,!and!exhibits!separate!
inhomogeneously!broadened!peaks!above!and!below!the!diagonal!at!Ωemis~16400!
cmV1.!!The!higher!energy!peak!shows!a!shoulder!on!the!low!energy!edge!suggesting!
the!presence!of!a!third!peak.!!

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!

3.6 104

3.6 10

4

b

a
3.5

3.5
-1

exc (cm )exc (cm-1)

3.4
3.3
3.2

3.4
3.3

3.1
3.2

3
2.9

1.5

1.6


emis

-1

(cm )

1.7

1.8
10 4

3.1
1.55

1.6

1.65
1.7

(cm-1)
emis

1.75
1.8
10 4

!
Figure(5.5.((Absolute!value!2D2Q!spectra!of!(a)!mPlum!and!(b)!cresyl!violet.!
!

To!better!understand!the!2D2Q!peak!structure!of!mPlum,!we!explored!the!

excitation!dependence!of!this!response.!!The!spectrum!collected!at!blue!excitation!
(16949!cmV1)!in!Figure!5.6b!displays!two!peaks!of!roughly!equal!intensity!vertically!
aligned!at!~17000!cmV1!closely!matching!the!linear!absorption!peak!of!16978!cmV1.!!
The!red!excitation!(Figure!5.6f,!15748!cmV1)!produces!a!strong!feature!above!the!
diagonal!with!Ωemis!=15500!cmV1,!along!with!a!weaker!peak!below!the!diagonal!at!
slightly!higher!energy.!!We!attribute!the!peak!above!the!diagonal!to!the!anomalous!
shoulder!observed!in!Figure!5.5.!!Moreover,!the!presence!of!a!third!response!
indicates!mPlum!demonstrates!an!electronic!structure!differing!from!that!of!cresyl!
violet,!which!was!well!modeled!as!a!3LS.!!The!orientation!and!eccentricity!of!this!
anomalous!peak!varies!slightly!from!those!of!blue!excitation!suggesting!unique!bath!
coupling!among!the!different!states.!!!!!

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!

3.8 104

3.8 10

a

3.2

1.5

1.6
1.7

(cm-1)

1.8emis

3.8 10

-1

3.4
3.2

1.5

1.6
1.7

(cm-1)

1.8

emis

4

3.8 10

d

3.2

2.8
1.4

1.9
10 4

4

3.8 10

e



3.2
3

1.6
1.7

(cm-1)

1.8

emis

1.9
10 4

-1

3.4
3.2

2.8
1.4

1.8

1.9
10 4

1.6
1.7

(cm-1)

1.8

1.9
10 4

4

f

3

1.5

1.6
1.7

(cm-1)

3.6
exc (cm )(cm-1)
exc

3.4

1.5

emis

3.6

2.8
1.4

3.4

3

2.8
1.4

1.9
10 4

3.6
(cm-1)

3.6

3

2.8
1.4

4

c

exc (cm )(cm-1)
exc

3.4

3

exc

3.8 10

3.6





exc

(cm-1)

3.6



4

b

3.4
3.2
3

1.5

1.6
1.7

(cm-1)
emis

1.8

1.9
10 4

2.8
1.4

1.5

emis

!

Figure(5.6.((Absolute!value!2D2Q!spectra!of!mPlum!with!excitation!center!
wavelengths!of!(a)!17452,!(b)!16949,!(c)!16447,!(d)!16207,!(e)!16000,!and!(f)!15748!
cmV1!all!with!a!bandwidth!of!1000!cmV1!+/V!3%.!!Measured!excitation!spectra!are!
overlaid!in!green!along!the!emission!frequency!axis.!
Model#Calculations#!

Simulations!of!2D2Q!spectra!were!performed!with!a!widelyVused!nonlinear!

response!function!approach!to!the!third!order!polarization187.!

P

(3)

∞ ∞ ∞

(r, t ) = ∫ ∫ ∫ dt3 dt2 dt1E (r, t − t1 ) E (r, t − t1 − t2 ) E (r, t − t1 − t2 − t3 ) R(3) (t1, t2 , t3 ) ! (5.1)!
−∞ −∞ −∞

The!following!Liouville!pathways!define!the!response!functions!for!a!NVlevel!
electronic!system78.!
3

4
N
!i$
*
S (t1, t2 , t3 ) = # & Θ (t1 ) Θ (t2 ) Θ (t3 ) ∑ ∑*+ Rαβ (t1, t2 , t3 ) − Rαβ(t1, t2, t3 ),- !
"!%α =1 β =1(3)!

(5.2)!

In!addition!to!the!3LS!response,!three!additional!electronic!structures!were!
explored:!!a!mixture!of!two!independent!3LS!and!with!the!effect!of!two!closely!

134!
!
spaced!levels!in!the!first!and!second!excited!states!(separation!~1500!cmV1),!
denoted!as!1V2V1!and!1V1V2!respectively!(Figure!5.7).!!

Independent 3LS

1-2-1

1-1-2

!

Figure(5.7.((Electronic!level!structures!considered!for!modeling!of!2D2Q!data:!!two!
independent!three!level!systems!and!1V2V1!and!1V1V2!four!level!systems.!!The!
arrows!represent!possible!responses!for!double!quantum!coherence!pathways.(
The!inclusion!of!additional!electronic!states!alters!the!response!expected!for!a!single!
3LS.!!The!doubleVsided!Feynman!diagrams!in!Figure!5.8!describe!these!additional!
responses!for!the!1V1V2!level!structure.!!Here,!R1!and!R2!represent!the!previously!
described!contributions!for!a!3LS187.!!!

R1

R2

R3

R4

0

0

1

1

0

0

1

1

1

0

2

1

1

0

3

1

t3

2

0

2

0

3

0

3

0

t2

1

0

1

0

1

0

1

0

t1

0

0

0

0

0

0

0

0

!
Figure(5.8.((LightVmatter!interactions!representing!the!double!quantum!responses!
of!a!1V1V2!electronic!level!structure.(!

To!simplify!spectral!interpretation,!calculations!were!performed!in!the!Bloch!

limit!with!an!exponential!dephasing!constant!Γij!and!impulsive!excitation.!!

135!
!
Transition!dipole!moments,!μij,!were!set!to!be!equal!between!all!states!and!an!
anharmonicity!of!1000!cmV1!was!assumed!for!all!calculations.!!Following!these!
approximations,!the!frequency!domain!response!for!a!3LS!following!the!above!
assumptions!within!the!rotating!wave!approximation!is!

S(!

2 D2 DQ−3LS )(Ωexc , Ωemis ) =

2
µ 21
µ102
×
2
(Ωexc − Ω20 ) + Γ 220%' Γ Γ − (Ω − Ω ) (Ω − Ω ) Γ Γ − (Ω − Ω ) (Ω − Ω ) )'
emis
10
exc
20
emis
21
exc
20
− 21 20& 10 20
*
2
2
2
2
'(
'+
(Ωemis − Ω10 ) + Γ10(Ωemis − Ω21 ) + Γ 21!

(5.3)! !

Here,!Ωexc!and!Ωemis!are!the!corresponding!frequency!analogues!of!t2!and!t3.!!
Moreover,!the!terms!inside!the!brackets!represent!the!R1!and!R2!responses!
described!by!the!Feynman!diagrams!in!Figure!5.8.!!A!treatment!including!nearly!
degenerate!states!at!either!the!first!or!second!excited!level!incorporates!additional!
responses!following!a!similar!pattern!as!the!3LS!case.!!For!1V1V2,!the!predicted!
response!is!as!follows!

S(

2 D2Q−112)(Ωexc , Ωemis ) =

2
µ 21
µ102
×
2
(Ωexc − Ω20 ) + Γ 220%' Γ Γ − (Ω − Ω ) (Ω − Ω ) Γ Γ − (Ω − Ω ) (Ω − Ω ) )'
emis
10
exc
20
emis
21
exc
20
− 21 20& 10 20*+2222(Ωemis − Ω10 ) + Γ10(Ωemis − Ω21 ) + Γ 21('+'

µ312µ102×2(Ωexc − Ω30 ) + Γ 302!

!

(5.4)!

%' Γ Γ − (Ω − Ω ) (Ω − Ω ) Γ Γ − (Ω − Ω ) (Ω − Ω ) )'
emis
10
exc
30
emis
31
exc
30
− 31 30& 10 30
*
2
2
2
2
'(
'+
(Ωemis − Ω10 ) + Γ10(Ωemis − Ω31 ) + Γ 31

with!two!additional!terms!stemming!from!simultaneous!coherence!within!the!nearly!
degenerate!doubly!excited!states.!!Likewise,!for!the!case!of!two!levels!in!the!singly!
excited!manifold,!the!signal!is!predicted!to!have!the!following!form.!!

136!
!
S(

2 D2Q−121)(Ωexc , Ωemis ) =2µ322 µ 20×2(Ωexc − Ω30 ) + Γ 302%' Γ Γ − (Ω − Ω ) (Ω − Ω ) Γ Γ − (Ω − Ω ) (Ω − Ω ) )'
emis
20
exc
30
emis
32
exc
30
− 32 30& 20 30
*+
2
2
2
2
'(
'+
(Ωemis − Ω20 ) + Γ 20
(Ωemis − Ω32 ) + Γ 32

µ312µ102×2(Ωexc − Ω30 ) + Γ 302!

!

(5.5)!

%' Γ Γ − (Ω − Ω ) (Ω − Ω ) Γ Γ − (Ω − Ω ) (Ω − Ω ) )'
emis
10
exc
30
emis
31
exc
30
− 31 30& 10 30*2222(Ωemis − Ω10 ) + Γ10(Ωemis − Ω31 ) + Γ 31('+'

The!4LS!responses!appear!similar!to!that!of!a!3LS!with!signal!arising!from!ground!to!
first!excited!state!and!first!to!doubly!excited!transitions!respectively!with!opposite!
signs.!!However,!the!additional!level!leads!to!a!second!pair!of!peaks!in!each!case!
compared!to!a!3LS.!!
Results#of#Model#Calculations#!

2D2Q!spectra!were!simulated!within!the!Bloch!limit!for!the!three!different!

models!of!the!electronic!structure!depicted!in!Figure!5.7.!!The!relative!
anharmonicity!of!the!level!positions!is!an!important!factor!when!interpreting!2D2Q!
spectra.!!We!considered!all!possible!permutations!for!all!level!schemes.!!These!are!
denoted!as![i#j],!where!i!and!j!are!the!anharmonicities!of!the!two!states!in!the!doubly!
excited!manifold.!!For!example,!the!1V1V2!electronic!structure!with![+!V]!
anharmonicity!has!one!state!at!less!than!double!the!value!of!the!single!quantum!
energy!(i.e.!“V“!anharmonicity)!and!one!state!at!more!than!double!the!value!of!the!
single!quantum!energy!gap!(i.e.!“+”!anharmonicity).!!Level!positions!were!chosen!to!
best!reproduce!the!experimental!data!and!exact!values!are!tabulated!below.!!!
(

137!
!
Table(5.1.!!Energy!values!(cmV1)!for!various!electronic!level!structures!used!to!
model!2D2Q!experimental!data.!!!!!
Two$3LS$&[+&4]&LA1=LA2&

&&

0&

0&

17000&

17000&

33000&

35000&&[+&4]&LA1≠LA2&0&0&16500&

17500&

32000&

36000&[+&+]&

0&0&17000&

17000&

35000&

36000&[4&4]&

0&0&17000&

17000&

32000&

33000&

&
&
&
&
&
&
&
&
&
&
&
&
&
&
&

1)1)2$&[+&4]&[+&+]&[4&4]&

0&0&0&17000&

17000&

17000&

33000&

35000&

32000&

35000&

36000&

33000&

1)2)1$
[+]&

[4]&

0&

0&

16500&

16500&

17500&

17500&

36000&

32000&

&&
&
&
&
&&

!
This!initial!effort!is!only!intended!to!demonstrate!the!unique!electronic!structure!of!
mPlum!relative!to!a!known!three!level!system.!!Several!factors!that!play!an!
important!role!in!determining!the!peak!shapes,!including!pulse!overlap!effects,!finite!
excitation!bandwidth,!and!a!realistic!systemVbath!coupling!model!are!neglected!here!
and!will!be!explored!in!a!future!report.!!

138!
!
!

Simulated!double!quantum!coherence!spectra!are!displayed!below.!!For!two!

independent!3LS,!there!are!three!possible!anharmonicity!configurations,![V!V],![+!V],!
and![+!+].!!Cases!with!an!equal!and!unequal!single!quantum!energy!gap!with![+!V]!
anharmonicity,!illustrated!in!Figure!5.9,!most!closely!match!the!experimental!data.!!
While!simulated!spectra!of![+!+]!and![V!V]!anharmonicities!are!shown!in!Figure!5.10.!

4 10

4

4 10

b

3.8

3.8

3.6

3.6

exc (cm-1)

exc (cm-1)

a

3.4
3.2

3.4
3.2

3

3

2.8

2.8

2.6

4

1.4

1.6


emis

(cm-1)

1.8

2

10 4

2.6

1.4

1.6


emis

(cm-1)

1.8

2
10 4

Figure(5.9.((Simulated!absolute!value!2D2Q!spectra!for!two!independent!3LS.!!The!
two!spectra!represent!cases!of!(a)!equal!and!(b)!unequal!linear!absorption!peaks!
with![+!V]!anharmonicity.(((
!
!
!

!

139!
!
4 10

4

4 10

b

3.8

3.8

3.6

3.6

exc (cm-1)

exc (cm-1)

a

3.4
3.2

3.4
3.2

3

3

2.8

2.8

2.6

4

1.4

1.6


emis

(cm-1)

1.8

2.6

2

10 4

1.4

1.6


emis

(cm-1)

1.8

2
10 4

Figure(5.10.((Simulated!absolute!2D2Q!spectra!of!two!3LS!with!equal!linear!
absorption!and!(a)!negative!and!(b)!positive!anharmonicity.!!!
For!the!1V1V2!electronic!structure,!there!are!three!possible!anharmonicities,![V!V],![+!V],!and![+!+].!!Simulated!spectra!for!the![+!V]!configuration!is!provided!in!Figure!5.11!
and!the!remaining!permutations!are!shown!in!Figure!5.12.!!!

4 10

4

3.8

(cm-1)

3.2



3.4

exc

3.6

3
2.8
2.6

1.4

1.6


emis

(cm-1)

1.8

2
10 4

!

Figure(5.11.((Simulated!absolute!value!2D2Q!response!of!the!1V1V2!level!structure!
with![+!V]!anharmonicity.!

!

140!
!
4 10

4

4 10

b

3.8

3.8

3.6

3.6

exc (cm-1)

exc (cm-1)

a

3.4
3.2

3.4
3.2

3

3

2.8

2.8

2.6

4

1.4

1.6


emis

(cm-1)

1.8

2.6

2

1.4

10 4

1.6


emis

(cm-1)

1.8

2
10 4

!

Figure(5.12.((Simulated!2D2Q!spectra!of!the!1V1V2!level!structure!with!(a)!negative!
and!(b)!positive!anharmonicity.(!
The!simulated![+!V]!spectrum!appears!almost!identical!to!two!independent!3LS!with!
equal!linear!absorption!positions!and![+!V]!anharmonicity.!!Lastly,!for!the!1V2V1!
electronic!structure,!there!are!two!combinations!considered!with![V!V]!and![+!+]!
anharmonicity.!

4 10

4

4 10

b

3.8

3.8

3.6

3.6
exc (cm-1)

exc (cm-1)

a

3.4
3.2

3.4
3.2

3

3

2.8

2.8

2.6

4

1.4

1.6


emis

(cm-1)

1.8

2

10 4

2.6

1.4

1.6


emis

(cm-1)

1.8

2
10 4

Figure(5.13.((Simulated!absolute!value!2D2Q!response!of!the!1V2V1!level!structure!
with!(a)!negative!and!(b)!positive!anharmonicity.!
The!modeled!spectra!are!most!conveniently!characterized!by!the!number!of!peaks!

!

141!
!
present!along!with!their!sign!and!relative!position!to!the!diagonal!boundary.!!Within!
these!constraints,!we!can!immediately!discount!a!number!of!possible!level!schemes,!
leaving!two!3LS![+!V]!and!1V1V2![+!V]!as!the!most!likely!candidates.!!We!further!
discriminate!between!these!models!in!the!Discussion!section.!!
2D2Q#Spectroscopy#of#mKate#Mutants#
To!explore!the!dependence!of!alteration!of!the!local!electrostatic!
environment,!2D2Q!spectra!were!measured!for!mKate!and!related!point!mutants.!!
Variation!at!position!158!in!mKate!and!its!ancestors!is!known!to!strongly!influence!
their!photophysical!properties!leading!to!a!9Vfold!improvement!in!photostability!in!
TagRFP!S158T!and!3Vfold!increase!in!brightness!in!mKate!S158A188V189.!!It!is!
reasonable!to!assume!that!the!alteration!of!the!chromophore!electronic!structure!
may!underlie!this!behavior.!!These!results!are!presented!in!Figure!5.14.!!!

3.6 104

3.6 104

3.6 10

b

3.4

3.4

3.4

3.3

3.2

3.2

exc (cm-1)

3.5(cm-1)

3.5

exc

3.5

3.3

3.3
3.2

3.1

3.1

3.1

3

3

3

2.9

1.5

1.6


emis

(cm-1)

1.7

1.8
10 4

2.9

4

c



exc (cm-1)

a

1.5

1.6


emis

(cm-1)

1.7

1.8
10 4

2.9

1.5

1.6


emis

(cm-1)

1.7

1.8
10 4

Figure(5.14.((Absolute!value!2D2Q!spectra!of!(a)!mKate,!(b)!mKate!S158A,!and!(c)!
mKate!S158C.!
The!absolute!value!2D2Q!spectra!reveal!a!peak!pattern!similar!to!that!
observed!for!mPlum.!!Two!peaks!roughly!vertically!aligned!at!equal!Ωemis!fall!above!
and!below!the!diagonal,!while!additional!features!are!observed!at!lower!Ωemis!above!
the!diagonal.!!This!suggests!that!its!electronic!structure!likely!follows!the!1V1V2!

!

142!
!
scheme!proposed!for!mPlum.!!The!relative!intensities!of!these!features!vary!among!
the!mutants.!!This!likely!reflects!incomplete!overlap!with!the!center!transition!
frequencies!within!a!finite!excitation!bandwidth!and!implies!that!the!positions!of!
these!states!shift!with!alteration!of!the!local!electrostatic!environment.!!However,!
considering!only!these!preliminary!results,!a!conclusive!relationship!between!their!
electronic!structure!and!photophysical!parameters!is!unclear.!!Further!study!
utilizing!broadband!excitation!would!enable!more!precise!quantification!of!the!exact!
positions!of!the!electronic!states!and!their!correlation!with!mutation.!!Moreover,!
development!of!a!systematic!panel!of!variants!with!known!photophysical!behavior!
(e.g.!dark!state!conversion,!quantum!yield)!might!help!better!make!this!connection.!!!
5.4(Discussion!!
!

2D2Q!spectra!reported!for!dyes!in!bulk!solvents!yield!two!peaks!of!opposite!

sign!separated!by!their!anharmonicity!according!to!the!3LS!responses!described!in!
Figure!5.3179,183.!!We!first!reproduced!these!measurements!to!demonstrate!the!
validity!of!our!experimental!technique.!!The!third!peak!in!the!mPlum!spectra!
suggests!a!more!complex!electronic!excited!state!landscape.!!To!explain!the!
additional!spectral!feature,!we!favor!the!model!with!two!closely!spaced!levels!in!the!
doubly!excited!level,!with!opposite!anharmonicity.!!This!preference!was!established!
on!the!basis!of!simulated!2D2Q!spectra,!excitation!dependent!2D2Q!spectra,!and!
spectrallyVresolved!transient!absorption!data.!!!!
!

Since!the!excitation!spectral!bandwidth!did!not!fully!span!the!spectral!region!

of!interest,!excitation!dependent!2D2Q!measurements!were!performed!to!enable!

143!
!
more!complete!identification!of!the!optical!response.!!The!spectral!features!
observed!with!blue!excitation,!above!and!below!the!diagonal!in!Figure!5.6!reflect!
ground!to!first!excited!state!transitions!associated!with!closely!spaced!levels!in!
either!the!single!or!doubly!excited!manifolds!given!their!close!alignment!along!Ωemis!
with!the!linear!absorption!peak!of!16978!cmV1.!!The!strongest!feature!under!red!
excitation!represents!an!independent!response!arising!from!the!|2><1|!coherence!
predicted!in!R2.!!In!total,!we!observe!3!inhomogeneously!broadened!peaks,!2!
centered!at!Ωemis≈Ω10!and!1!below!the!diagonal!at!lower!Ωemis.!!Model!calculations!of!
the!two!3LS!and!1V1V2!level!structures!yield!responses!similar!to!the!experimental!
results.!!Simulated!spectra!in!the!Bloch!limit!show!a!total!of!4!peaks,!with!2!
vertically!aligned!at!Ωemis=Ω10!attributed!to!the!ground!to!first!excited!coherences!
and!1!each!at!lower!and!higher!Ωemis!representing!coherences!between!the!first!and!
doubly!excited!states!at!negative!and!positive!anharmonicity!respectively.!!!
!

Next,!we!considered!these!two!possible!models!within!the!context!of!RFP!

molecular!structure.!!A!model!with!two!3LS!implies!that!mPlum!is!a!mixture!of!two!
discrete!subensembles!likely!arising!from!some!structural!rearrangement!within!the!
local!protein!environment!or!chromophore!itself.!!While!cisVtrans!isomerization!has!
been!reported!for!some!RFP!variants,!these!interconversions!typically!lead!to!a!
significant!spectral!shift!in!the!linear!absorption!spectrum!following!from!disruption!
of!the!!"conjugated"system171,49C50,"189.""For"example,"the"spectral"shift"between"the"two"chromophore"isomers"leading"to"the"spectral"shift"of"mKate"relative"to"its"parent"TagRFP"is"~"900"cmC1.""The"linear"absorption"spectrum"of"the"lowest"energy"transition"in"mPlum"exhibits"a"single"broad"peak"(~2000"cmC1"FWHM)"with"a"144!
!
shoulder"at"higher"energy,"typical"of"RFPs"and"of"similar"width"to"the"peaks"
observed"in"the"2D2Q"spectra.""We"cannot"completely"discount"the"existence"of"conformers"with"smaller"spectral"shifts,"however,"experimental"attempts"to"date"addressing"such"conformational"heterogeneity"in"biological"systems"are"unclear"and"open"to"interpretation144.""Moreover,"one"would"not"expect"large"perturbations"at"the"doubly"excited"level"in"this"scenario.""These!results!suggest!mPlum!is!most!
consistent!with!a!1V1V2![+!V]!electronic!structure.!!This!electronic!structure!is!
consistent!with!SRTA!results!reported!in!Figure!5.3,!which!reveals!ESA!on!both!the!
red!and!blue!extremes!of!the!GSB.!!
!

Our!model!calculations!for!the!1V1V2![+!V]!level!scheme!predict!a!4th!peak!lying!

below!the!diagonal!at!Ωemis>!Ω10!corresponding!to!the!excited!state!response!from!
the!state!of!positive!anharmonicity.!!However,!this!feature!is!not!observed!in!the!
experimental!2D2Q!spectra.!!Although!we!do!not!have!a!quantitative!explanation!for!
the!absence!of!this!peak,!we!consider!three!possible!explanations.!!First,!we!discount!
the!possibility!that!the!peak!is!unobserved!due!to!insufficient!laser!spectral!
bandwidth.!!The!excitation!dependence!of!the!mPlum!2D2Q!spectrum!was!probed!
out!to!18500!cmV1!as!illustrated!in!Figure!5.6.!!The!short!wavelength!limit!is!dictated!
by!the!JILA!MONSTR!output!dichroic!mirror!that!separates!the!reference!laser!
(18797!cmV1)!from!the!femtosecond!excitation!beams.!!We!feel!this!spectral!cutoff!is!
unlikely!to!account!for!the!missing!peak!because!the!observed!peak!intensity!decays!
to!zero!at!17500!cmV1!along!Ωemis,!while!some!fraction!of!the!signal!is!still!observed!
above!the!diagonal!along!Ωexc.!!Since!the!position!of!this!positive!feature!is!coupled!
to!that!of!the!missing!peak,!it!seems!the!spectral!coverage!is!sufficient!to!observe!a!

145!
!
similar!fraction!of!the!signal!below!the!diagonal.!!A!second!possible!explanation!is!
that!the!doubly!excited!mPlum!chromophore!undergoes!population!transfer!to!a!
neighboring!tryptophan!sidechain.!!Typically,!the!S1!absorption!band!of!tryptophan!
is!found!at!280!nm!(35714!cmV1),!which!coincides!with!the!edge!of!the!observed!
2D2Q!peaks!along!Ωemis.!!We!hypothesize!that!the!double!quantum!coherent!state!of!
the!chromophore!can!be!electronically!coupled!to!a!nearby!tryptophan,!which!then!
undergoes!radiative!deactivation,!and!therefore!does!not!contribute!to!the!nonlinear!
signal.!!Crystal!structures!of!mPlum!reveal!three!tryptophan!residues!within!5!Å!of!
the!central!chromophore!(PDB!2QLG).!!A!third!scenario!invokes!an!explanation!put!
forward!by!Kim!et#al.!to!explain!the!mismatch!in!intensity!of!aboveV!and!belowV
diagonal!features!in!2D2Q!spectra!of!cresyl!violet190.!!They!attribute!this!to!different!
dephasing!rates!of!the!two!coherence!states!in!the!last!evolution!period,!which!ends!
in!emission!of!the!signal.!!Following!this!rationale!for!a!1V1V2!electronic!structure,!
we!propose!that!Γ31>!Γ21,!thus!reducing!its!relative!contribution!in!the!2D2Q!
spectrum.!!One!possible!reason!for!the!short!dephasing!time!Γ31!is!a!fast!
nonradiative!relaxation!from!state!|3>!to!|2>,!resulting!from!the!small!energy!gap.!!
This!effect!would!decrease!the!contribution!of!R4!relative!to!R2.!!!
!

Other!factors!considered!to!explain!the!unique!2D2Q!response!of!mPlum!are!

vibronic!transitions!and!finite!pulse!effects.!!As!discussed!and!modeled!by!Nemeth!et#al.,!distortions!arising!from!temporal!pulse!structure!can!lead!to!additional!peaks!
and!sometimes!obscure!spectral!assignment179.!!However,!these!features!were!
found!to!appear!along!the!antidiagonal!direction!in!their!simulated!spectra.!!The!
extraneous!feature!in!our!results!falls!within!the!area!predicted!for!a!degenerate!

146!
!
doubly!excited!electronic!manifold.!!For!a!vibronic!response,!one!would!expect!
peaks!shifted!to!lower!energies!along!the!Ωemis!dimension!as!previously!modeled190.!!!!
!

These!experiments!provide!a!means!of!contrasting!the!information!extracted!

from!several!frequently!employed!nonlinear!techniques!including!transient!
absorption,!transient!grating,!and!multidimensional!methods.!!In!a!previous!report,!
we!identified!an!ESA!component!to!the!red!of!GSB!via!SRTG!spectroscopy168.!!
Moreover,!as!illustrated!in!Figure!5.3,!excitation!dependent!SRTA!spectra!show!ESA!
bounding!GSB!at!both!higher!and!lower!energies.!!While!the!1Q2D!spectra!of!mPlum!
and!cresyl!violet!exhibit!a!single!inhomogeneously!broadened!peak,!the!2D2Q!data!
suggest!a!more!complicated!excited!state!landscape!as!illustrated!below.!
4

4

1.8

1.8

1.8

1.6
1.5
1.4
1.4

-1

1.7

exc

1.7

exc (cm )1.9 10t2=75 ps

(cm-1)

1.9 10t2=10 psexc (cm-1)

4

1.9 10
t2=0

1.6
1.5

1.5

1.6
1.7
emis (cm-1)

1.8

1.6
1.5

1.4
1.4

1.9
10 4

1.7

1.5

1.6
1.7
emis (cm-1)

1.8

1.4
1.4

1.9
10 4

1.5

1.6
1.7
emis (cm-1)

1.8

1.9
10 4

!

Figure(5.15.((mPlum!absolute!value!2D1Q!spectra!at!t2=0,!10,!and!75!ps.!!!
4

4

1.8

1.8

1.8

exc

1.6
1.5
1.4
1.4

1.7

-1

1.7

exc (cm )1.9 10t2=400 fs(cm-1)

1.9 10t2=150 fs





exc

(cm-1)

4

1.9 10
t2=0

1.6
1.5

1.5

1.6
1.7
emis (cm-1)

1.8

1.9
10 4

1.4
1.4

1.7
1.6
1.5

1.5

1.6
1.7
emis (cm-1)

1.8

1.9
10 4

1.4
1.4

1.5

1.6
1.7
emis (cm-1)

1.8

1.9
10 4

!

Figure(5.16.((Cresyl!violet!absolute!value!2D1Q!spectra!at!t2=0,!150,!and!400!fs.((!
For!instance,!it!would!be!infeasible!to!differentiate!a!single!broad!ESA!contribution!

147!
!
from!two!separate!ESA!contributions!considering!the!SRTA!results!alone.!!The!2D2Q!
spectra!facilitate!this!distinction!and!demonstrate!the!power!of!multidimensional!
techniques!in!unraveling!congested!spectral!features.!
!

To!date,!most!insight!into!RFP!electronic!structure!has!originated!largely!from!

theoretical!model!calculations191V192.!!For!example,!DFT!calculations!demonstrated!
the!environmental!influence!of!tuning!1PA!and!2PA!properties!in!the!progenitor!
DsRed148.!!In!another!study,!the!prospect!of!!V!!stacking!was!explored!as!a!means!of!
generating!GFP!variants!with!redVshifted!emission174.!!In!contrast,!experimental!
accounts!addressing!the!energies!and!photophysics!of!higher!excited!states!in!RFPs!
are!sparse.!!As!mentioned!above,!2PA!investigations!on!the!mFruits!(mPlum!
included)!revealed!absorption!on!the!blue!edge!near!300!nm175.!!This!would!
coincide!with!the!doubly!excited!manifold!of!mPlum,!although,!they!were!explained!
as!a!resonance!enhancement!effect!resulting!from!overlap!of!the!excitation!laser!
frequency!and!singly!excited!state.!!Since!2D2Q!is!not!sensitive!to!this!resonance!
enhancement!phenomenon,!our!results!represent!a!new!response!independent!from!
the!2PA!spectra.!!It!may!also!explain!why!cross!sections!vary!widely!in!this!region!
across!the!mFruit!family.!!!
!

The!finding!of!multiple!doublyVexcited!states!in!FPs!may!have!implications!for!

the!ongoing!development!of!new!fluorophores!and!imaging!techniques.!!It!would!be!
useful!to!consider!the!likelihood!of!excitedVstate!absorption!as!a!criterion!for!choice!
of!FP!variants!in!a!particular!imaging!modality.!!SiteVdirected!mutagenesis!has!
shown!that!FP!photostability!and!brightness!vary!widely!with!a!mere!single!

148!
!
modification!near!the!chromophore146.!!2D2Q!may!be!helpful!in!evaluating!potential!
candidates.!!As!described!above!(Equations!5.3V5.5),!the!magnitude!of!the!double!
quantum!response!is!dependent!on!the!individual!strengths!of!the!constituent!single!
quantum!transitions.!!Thus,!direct!quantification!of!the!Ω20!transition!strength!is!not!
possible!with!2D2Q!experiments!alone.!!However,!this!technique!may!be!used!to!
precisely!survey!the!landscape!of!electronic!excited!states.!!Broadband!(i.e.!
exceeding!2000!cmV1!spectral!width)!2D2Q!would!facilitate!observation!of!exact!
level!positions.!!These!studies!will!be!particularly!relevant!for!evaluating!
photoswitchable!variants!of!FPs!for!nanoscopic!imaging!methods!that!rely!on!
excitation!intensities!of!10V100!kW/cm2!or!higher43,!193.!!In!this!intensity!regime,!
multiVphoton!absorption!is!possible!even!with!cw!excitation,!and!the!probability!of!
photobleaching!due!to!multiple!mechanisms!increases!relative!to!lowerVintensity!
excitation!characteristic!of!widefield!illumination!(<!1!kW/cm2).!Thus,!tuning!of!
excitation!wavelength!to!minimize!absorption!into!doubleVquantum!states!could!
result!in!substantial!improvement!of!photostability.!!!
5.5(Conclusions(!

2D2Q!measurements!indicate!that!mPlum!displays!an!electronic!structure!that!

differs!significantly!from!a!simple!organic!dye!in!solution,!cresyl!violet!in!methanol.!!
We!attribute!this!response!to!closely!spaced!levels!in!the!doubly!excited!manifold!
with!opposite!anharmonicity.!!These!conclusions!are!supported!by!excitation!
dependent!SRTA!data!and!simulated!spectra!derived!from!third!order!nonlinear!
response!functions.!!Since!they!share!virtually!the!same!chromophore!structure,!we!
expect!that!this!electronic!landscape!to!be!broadly!representative!of!all!red!

149!
!
fluorescent!proteins,!though!the!positions!of!the!levels!are!likely!sensitive!to!the!
local!chromophore!environment.!!These!results!shed!light!on!the!largely!unexplored!
electronic!structure!of!RFPs!as!well!as!demonstrate!the!power!of!multidimensional!
spectroscopy!in!unraveling!congested!spectral!features.!!(
!
!
!
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(

150!
!
Chapter(VI!
Conclusions(and(Future(Outlook!
!
6.1(Future(Outlook(!

It!is!important!to!place!this!work!in!the!context!of!the!previous!fluorescent!

protein!research.!!Work!carried!out!on!red!fluorescent!proteins!to!date!has!been!
primarily!driven!by!an!effort!to!optimize!their!emission!wavelength,!fluorescence!
quantum!yield,!and!photostability.!!Development!of!new!FPs!was!largely!
accomplished!via!the!notion!of!“rational!design”!stemming!from!the!information!
derived!from!the!standard!methods!of!structural!biology!and!protein!engineering!
(e.g.!xVray!crystallography,!linear!absorption!and!emission!spectroscopy,!and!siteV
directed!mutagenesis).!!These!methods,!while!useful!for!obtaining!a!steadyVstate!
picture!of!protein!systems,!are!insensitive!to!the!inherent!dynamic!aspects!of!these!
complex!molecular!systems.!!In!this!thesis,!sophisticated!nonlinear!spectroscopic!
techniques!were!utilized!to!provide!new!insight!into!the!origin!of!RFP!photophysical!
properties.!!These!results!indicate!that!a!dynamic!perspective!is!necessary!to!gain!a!
deeper!understanding!of!RFP!photophysics!and!may!help!guide!the!engineering!of!
nextVgeneration!derivatives.!!!
!

Adequate!computational!modeling!is!critical!for!analysis!of!transient!

spectroscopic!data.!!The!transient!grating!results!presented!in!Chapters!III!and!IV!
were!interpreted!with!the!aid!of!classical!molecular!dynamics!simulations.!!Such!
treatment!provides!quantification!of!only!ground!state!molecular!fluctuations!and!is!
approximated!to!represent!excited!state!relaxation!within!linear!response!theory.!!A!

151!
!
more!accurate!simulation!of!excited!state!dynamics!could!be!achieved!by!employing!
a!quantum!mechanical!modeling!of!the!chromophore!electronic!structure.!!Hybrid!
transient!quantum!mechanicalVmolecular!mechanics!(QM/MM)!methods!have!been!
developed!to!simulate!dynamics!of!complex!biological!systems!such!as!proteins!and!
nucleic!acids194.!!For!applications!involving!an!optical!probe!buried!in!a!protein,!a!
typical!strategy!is!to!treat!the!chromophore!and!important!sidechain!residues!
quantum!mechanically,!while!motions!of!the!remaining!residues!and!solvent!
molecules!are!approximated!with!a!classical!force!field.!!This!approach!enables!a!
more!accurate!quantification!of!the!environmental!interactions!with!electronically!
excited!probe,!which!is!critical!for!developing!a!fundamental!molecular!
understanding!of!solvation!response.!!The!conclusions!drawn!in!Chapter!III,!namely,!
the!hydrogen!bond!switching!at!the!chromophore!acylimine!carbonyl!may!be!
further!refined!with!QM/MM!simulations!to!pinpoint!specific!vibrational!modes!
integral!for!this!phenomenon!and!its!role!in!extending!the!Stokes!shift!of!mPlum!and!
TagRFP675.!!!
!

The!2D2Q!study!described!in!Chapter!V!demonstrated!the!power!of!

multidimensional!spectroscopy!in!unraveling!congested!spectral!features!of!
condensed!phase!electronic!lineshapes.!!In!particular,!a!proposed!electronic!
structure!was!deduced!given!the!number!and!pattern!of!peaks!observed!in!modeled!
spectra.!!Further!understanding!of!these!results!could!be!achieved!through!various!
experimental!and!theoretical!improvements.!!First,!excitation!with!pulses!of!limited!
spectral!bandwidth!yields!only!partial!coverage!of!the!electronic!landscape!and!
hinders!spectral!assignment.!!Simultaneous!resolution!of!all!nonlinear!responses!

152!
!
would!be!possible!with!broadband!excitation!and!greatly!simplify!the!task!of!
assigning!multiple!overlapping!spectral!features.!!Second,!detection!of!the!signal!
phase!would!also!enhance!clarity!of!the!spectral!features!enabling!determination!of!
the!sign!of!constituent!peaks.!!These!refinements!are!possible!with!only!relatively!
minor!tweaks!to!the!current!experimental!platform!(JILA!MONSTR).!!Finally,!
incorporation!of!a!more!rigorous!modeling!effort!would!enable!accurate!
determination!of!important!variables!such!as!peak!position,!transition!strength,!and!
vibronic!coupling.!!This!could!be!accomplished!through!application!of!a!multimode!
Brownian!Oscillator!model,!the!current!standard!treatment!used!to!characterize!
systemVbath!coupling!in!the!condensed!phase.!!These!improvements!applied!to!the!
study!of!point!mutants!that!are!known!to!strongly!influence!RFP!photophysics!(e.g.!
mKate!S158!series)!might!help!connect!proteinVchromophore!interactions!to!their!
excited!electronic!structure!and!ultimate!spectral!properties.!!Such!information!
would!be!useful!for!the!ongoing!effort!to!design!more!robust!RFP!variants.!!!!
(
(
(
(
(
(
(
(
(
(

153!
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!

165!
!
Appendix(!
A.!!Data(Acquisition(Software!
Labview#Software#Programs!
2DFTS_scan_DQC.vi!
MoveStages.vi!
View!CCD.vi!
TGVFROG.vi!
Collect!Mightex!image!and!bin.vi!
pulse!overlap!webcam!Mightex.vi!
PumpProbeDiagnostics.vi!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!

166!
!
B.((Data(Processing(Software(
Matlab!(Mathworks)!was!the!primary!software!program!used!to!process!
experimental!data.!!Several!scripts!were!written!for!each!individual!experiment.!!
The!mathematical!conversions!within!these!scripts!are!represented!in!the!flow!
charts!below.!!Note:!!Since!the!functional!forms!evolved!slightly!over!the!course!of!
this!project!and!the!names!were!not!chronological,!the!most!recent!versions,!and!
presumably!most!comprehensive,!are!documented!here.!!!
Transient#Grating#FROG#
Load Raw Data

PrepTGFROGdata.m[NRspecfile(i)]Load Time Delays[delay.dat]

Save Appended Data
[SI0total.dat]

Figure 1: Plot FROG

Extract Frequency Maxima

Figure 2: Plot Freq Maxima

Integrate along Frequency

Figure 3: Plot Autocorrelation

Calculate Spectral andTemporal Widths

!
!

167!
!
#
#
#
Spectrally#Resolved#Transient#Grating#Load Delay Times

PrepSRTGmatrix.m[Apoptimes.dat]

Load Raw Data
[spec(i).dat]

Subtract Background

Plot Emission Slice
Plot Absorption Slice
Plot Raw Spectral Data

Save Raw TG Data
[rawTG.dat]

Save Wavelength Vector[wave.dat]

Save Delay Times[delay.dat]

Integrate along Frequency

Plot Integrated TG Data

Save Integrated TG Data
[intTG.dat]

!

168!
!

Load Integrated TG Data
[intTG.dat]

averageSRTGdata.m

Load Raw TG Data
[rawTG.dat]

Load Delay Times[delay.dat]

Load Wavelengths[wave.dat]

Normalize to T9

Plot Early TG Data
Plot Late TG Data
Plot Integrated TG Data
Plot Maxima

Plot TG Slices at Ti
Plot Normalized TG Slices at Ti

Save Integrated TG Data
[intTG.dat]

Save Averaged TG Data
[TGavg.dat]

Save Delay Times[delaylog.dat]

!

169!
!
!

Load TG Data
[TGavg.dat]

SRTGfit.m

Load Delay Times[delay.dat]

Load Wavelengths[wave.dat]

Specify Range

Conduct Peak Fitting[peakfit.m]

Plot GSB Peak Centers
Plot SE Peak Centers
Plot Example Fits
Plot RMS Error

Save GSB Peak Centers[blcp.dat]

Save SE Peak Centers[secp.dat]

Save RMS Error[rmserr.dat]

Save Fit Delay Times[delay.dat]

!
#

170!
!
#
2D#Fourier#Transform#Spectroscopy#
Load Reference Spectrum
REF00.dat

Prep2DFTSdata.m

Calculate Frequency Axis
emisfreqTHzRaw.dat
emisfreqWavnRaw.dat

Load Rephasing/Nonrephasing Data
Rspec(i)NRspec(i)Assemble Data into Matrix

Divde spectra by sqrt(reference)

Figure 1: Plot FROG
Plot Emission Slice
Plot Excitation Slice

Save Spectral Data
SI0total.dat

!
!

171!
!

Select Input Parameters
Step size# of contours
Undersampling ratio
Double quantum scan?
Frequency range
Emission time cut interval

generateDQC.m

Load Spectral Data
SI0total.dat

Resample emission dimension into THz
resampleWL2freq.m

Save Ωemis vector
emisFreqWavn.dat
emisFreqTHz.dat

Spectrally filter along Ωemis

Figure 1: Plot Time Cut Slice
Figure 2: Image Tau-t data before/after filtering
Figure 3: Image Omega_emis-tau data
Figure 4: Plot FWM integrated signal versus tau

Calculate Phase Corrected Data

Figure 5: Plot slice versus emission freq
Plot extracted phase versus emission freq

Fourier transform data along τ to obtain Ωabs
Zero pad to nearest multiple of 2
Convert rephasing and nonrephasing separately

Calculate Ωabs axis
Select Under/oversampled
Save Ωabs vector
absFreqWavn.dat
absFreqTHz.dat

Resample to desired frequency range
interp2.m

Figure 6: Contour plot absolute value spectrum
Figure 7: Contour plot complex spectra

Save absolute value, real, and imaginary spectral data
absFTS.dat
realFTS.dat
imagFTS.dat

!

172!
!
C.((Data(Modeling(Software(Spectron#2.8#
The!modeled!2Q2D!data!presented!in!Chapter!V!were!calculated!using!the!
Spectron!2.8!software!program.!!This!program!was!developed!by!the!research!group!
of!Shaul!Mukamel!with!the!intent!of!providing!a!universal!package!for!conducting!
calculations!of!optical!nonlinear!spectroscopic!signals!of!arbitrary!systems.!!!
(D.((JILA(MONSTR(Calibration(Procedures(
The!JILA!Multdimensional!Optical!Nonlinear!SpecTRometer!(MONSTR)!was!
developed!as!a!versatile!platform!for!conducting!nonlinear!optical!spectroscopy106.!!
A!detailed!summary!is!provided!in!the!reference!provided,!but!the!key!features!
together!with!practical!advice!for!its!routine!operation!will!be!discussed!in!this!
section.!!A!single!input!beam!is!divided!in!4!equal!parts!and!exit!the!device!in!the!
traditional!BOXCARs!geometry.!!Pulse!time!delay!is!controlled!with!independent!
direct!drive!linear!stages!with!5!cm!length!X,!Y,!and!Z!and!20!cm!for!the!U!axis.!!
These!lengths!translate!into!time!ranges!of!334!ps!and!1.3!nanoseconds!respectively.!!
Active!phase!stabilization!is!carried!out!by!monitoring!error!signals!generated!from!
a!copropagating!532!nm!cw!laser.!!Interference!patterns!from!the!top,!bottom,!and!
inter!decks!result!from!retroreflection!of!the!cw!light!off!the!output!dichroic!mirror!
and!are!routed!on!a!unique!path!to!independent!silicon!photodetectors.!!Error!
signals!are!sent!through!loop!filters!driving!a!piezoVmounted!mirror!within!each!
respective!beam!path.!!Subcycle!phase!corrections!are!conducted!in#situ#by!locking!
to!an!established!baseline.!!!

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Internal#Alignment#Internal!MONSTR!alignment!was!carried!out!as!suggested!by!Bristow!et#al.!in!
the!original!report106.!!A!cw!laser!was!used!at!low!intensity!as!an!alignment!
reference.!!For!initial!alignment,!the!top!and!bottom!halves!were!first!aligned!
separately!using!the!prearranged!apertures!ensuring!no!beam!drift!for!the!entire!
range!of!stage!movement.!!As!an!aside,!it!was!discovered!that!this!point!stability!
worsened!when!the!stages!were!physically!bolted!to!the!aluminum!case.!!As!such,!
the!U!stage!was!unbolted!and!wedged!along!its!aligned!pins!using!a!thin!rubber!
gasket.!!This!is!critical!since!k3!was!typically!scanned!through!its!entire!delay!range!
and!most!susceptible!to!alignment!drift.!(These!pieces!were!then!assembled!and!a!
minor!tweak!was!typically!necessary!to!account!for!slight!horizontal!offset!between!
the!top!and!bottom!decks.!!This!was!adjustment!was!made!with!a!small!change!of!
the!input!periscope.!!Upon!assembly!and!ensuring!point!stability,!the!output!beams!
were!propagated!a!large!distance!(several!meters)!to!examine!their!parallelism!and!
proximity.!!Minor!adjustments!to!the!final!mirror!along!each!beam!path!were!made!
to!achieve!the!necessary!1”!box!pattern.!!!
The!beam!overlap!at!the!sample!position!was!tested!two!different!ways.!!In!
one!method,!the!beams!were!aligned!through!a!µm!50!pinhole!and!small!tweaks!
were!made!to!the!output!mirrors!if!necessary.!!Alternatively,!a!surface!
backreflection!off!an!uncoated!flat!1”!diameter!fused!silica!window!placed!
perpendicular!across!the!converging!beams.!!This!reflection!was!focused!into!a!40x!
microscope!objective!(Edmund!NT43V908),!collimated,!and!imaged!with!a!CMOS!

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camera!(Mightex!S!series).!!This!represents!a!replica!of!the!focal!plane!at!the!sample!
position!and!served!as!a!convenient!aid!for!monitoring!beam!overlap.
Daily#Input#Alignment#
The!input!femtosecond!light!is!aligned!through!two!apertures!set!to!the!
predetermined!alignment!path!prior!to!the!MONSTR!input.!!It!was!found!that!
routine!alignment!to!these!references!with!external!steering!mirrors!yielded!
reproducible!beam!overlap!at!the!sample!position.!!This!was!verified!by!testing!the!
overlap!through!a!50!µm!pinhole.!!A!typical!beam!overlap!measured!at!the!replica!
focus!position!is!illustrated!in!Figure!A1.!

100
200
300
Pixel

400
500
600
700
800
900
1000
200

400

600 800
Pixel

1000 1200!

Figure(A1.(!Overlap!of!XYU!beams!at!the!replica!focus!position.!!!
Pulse#Time#Zero#Calibration#
Pulse!timing!at!the!sample!position!was!typically!established!in!two!steps.!!
First,!a!rough!search!for!time!zero!positions!was!conducted!by!monitoring!the!field!

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!
correlations!on!the!replica!focus!image!described!above.!!These!scans!were!
performed!and!analyzed!with!the!“pulse!overlap!webcam!Mightex.vi”!program.!!
Upon!measuring!the!approximate!relative!temporal!position!of!all!pulses,!a!more!
precise!measurement!was!made!at!the!sample!position.!!For!this!calibration,!three!
excitation!beams!(X,!Y,!U)!irradiated!a!sample!of!benzene!generating!a!nonresonant!
FWM!signal!in!the!k1Vk2+k3!direction.!!Benzene!was!chosen!given!its!high!nonlinear!
susceptibility!and!it!was!found!to!exhibit!no!interfering!resonant!or!nonresonant!
solvation!response.!!Iterative!scans!of!a!single!beam!were!performed!(TGVFROG.vi)!
with!subsequent!timing!adjustments!based!on!the!fitted!peak!of!the!FWM!signal!
integrated!along!detection!frequency.!!Representative!FROG!traces!and!fits!are!
presented!below!in!Figure!A2.!

aWavelength (nm)

700

1

b

Data
Fit

0.8
650
0.6
600

0.4
0.2

550
−60

−40

−20

0
20
Delay (fs)

40

60

0
-60

-40

-20

0
20
Delay (fs)40

60

Figure(A2.(!Representative!(a)!TG!FROG!spectrum!and!(b)!fitted!intensity!
autocorrelation!used!to!calibrate!timing!of!the!excitation!pulses.!
An!alternative!timing!procedure!could!be!conducted!using!the!pairwise!field!
correlations!at!the!replica!focus!position.!!Images!were!collected!as!a!function!of!
delay,!binned,!and!saved!(pulse!overlap!webcam!Mightex.vi).!!The!integrated!

!

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!
intensity!provides!a!means!of!rough!autocorrelation!as!well!as!relative!timing!of!the!
excitation!pulses.!!!
Timing!of!the!local!oscillator!reference!beam!(Z)!was!complicated!for!a!couple!
reasons.!!First,!attenuation!with!a!neutral!density!filter!was!necessary!to!obtain!an!
ILO:IFWM!ratio!of!~4:1.!!This!intensity!difference!made!for!difficult!detection!at!the!
replica!focus!position.!!As!such,!its!timing!was!established!by!autocorrelation!at!the!
sample!position.!!This!was!executed!with!either!a!twoVphoton!photodiode!(SiC)!or!
second!harmonic!generation!(BBO).!!Given!the!LO!delay!is,!in!principle,!arbitrary!for!
this!experimentation,!these!methods!were!found!to!produce!reasonably!accurate!
results.!!
Phase#Stabilization#Scheme#
Active!phase!stabilization!is!carried!out!by!monitoring!error!signals!
generated!from!a!copropagating!532!nm!cw!laser.!!Interference!patterns!from!the!
top,!bottom,!and!inter!decks!result!from!retroreflection!of!the!cw!light!off!the!output!
dichroic!mirror!and!are!routed!on!a!unique!path!to!independent!silicon!
photodetectors.!!Error!signals!are!sent!through!loop!filters!driving!a!piezoVmounted!
mirror!within!each!respective!beam!path.!!Subcycle!phase!corrections!are!conducted!
in#situ#by!locking!to!a!preestablished!DC!baseline.!!!
E.((Spectrally(Resolved(Signal(Detection(and(CCD(Calibration(Procedures(
Detection#Scheme!
The!spectrally!resolved!detection!scheme!can!be!summarized!as!follows.!!The!
nonlinear!signal!field!was!coupled!through!free!space!into!a!spectrometer!for!

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!
detection!(Princeton!Acton!SpectraPro!2300i).!!In!short,!a!10!cm!focal!length!PCX!
lens!focused!the!light!onto!the!entry!slit.!!It!was!then!dispersed!with!a!300!grv/mm!
diffraction!grating!blazed!for!600!nm,!collimated,!and!the!first!order!component!
focused!onto!a!liquid!N2Vcooled!1024!x!256!pixel!silicon!CCD!camera!(Roper!
Scientific).!!For!initial!alignment,!the!axial!position!and!rotation!of!the!camera!was!
adjusted!to!ensure!the!active!element!fell!in!the!correct!focal!plane!with!the!proper!
orientation.!!!
CCD#Calibration#Procedure!
Wavelength!calibration!of!the!CCD!camera!was!carried!out!using!the!
characteristic!emission!lines!of!a!mercuryVargon!lamp!(Oriel)!together!with!the!
known!output!of!our!532!nm!reference!laser!(Coherent!Verdi).!!For!a!typical!
measurement,!lamp!output!was!aligned!into!the!spectrometer!to!produce!sufficient!
signal!above!the!dark!count.!!Several!emission!lines!were!usually!visible!and!no!
fewer!than!4!spanning!the!spectral!region!of!interest!were!fit!versus!pixel!position!
assuming!linear!dependence.!!A!small!residual!of!the!532!nm!reference!laser!is!
transmitted!through!the!output!dichroic!of!the!JILA!MONSTR.!!The!local!oscillator!
beam!was!aligned!to!the!spectrometer!and!served!as!a!secondary!reference!used!for!
calibration.!!!!
F.((Additional(Experimental(Considerations(
Resolution#of#the#Stimulated#Emission#Band!
Acquisition!of!high!quality!SRTG!data!and!subsequent!interpretation!of!
solvation!dynamics!requires!simultaneous!resolution!of!the!Ground!State!Bleach!
(GSB)!and!Stimulated!Emission!(SE)!responses!within!a!finite!excitation!bandwidth!

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!
window.!!For!fluorescent!proteins,!only!species!with!a!large!Stokes!shift!(>50!nm)!
exhibited!these!two!components!without!ambiguity,!but!is!complicated!by!the!
presence!of!an!overlapping!Excited!State!Absorption!(ESA)!band.!!The!steady!state!
absorption!and!emission!spectra!of!mPlum!is!overlaid!with!its!SRTG!response!in!
Figure!A3!below.!!!

1

Absorption
Emission
SRTG

0.8
0.6
0.4
0.2
0
550

600
650
Wavelength (nm)700!

Figure(A3.!!mPlum!linear!absorption!and!emission!spectra!overlaid!with!its!SRTG!
response.!!!
Another!consideration!to!this!end!is!the!center!position!and!width!of!the!
excitation!source.!!Figure!A4!presents!SRTG!spectra!for!mPlum!E16Q!under!varying!
excitation!conditions.!!It!is!clear!that!extraction!of!the!excited!state!solvation!
dynamics!is!easiest!with!the!reddest!excitation,!which!provides!clearest!
differentiation!of!the!GSB!and!SE!contributions.!!On!the!other!hand,!given!a!finite!
excitation!bandwidth,!we!risk!sacrificing!resolving!dynamics!of!subensembles!at!the!

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!
blue!edge!of!our!laser!spectrum!in!cases!of!large!inhomogeneous!broadening.!!For!
these!experiments,!we!assume!roughly!constant!disorder!among!all!point!mutants!
given!the!minor!structural!perturbation!and!insensitivity!of!absorption!spectrum!
width.!

a

b

0.6

0.4

0.2

1

Normalized Amplitude (a.u.)

0.8

0.8

0.6

0.4

0.2

0

550

c

1

Normalized Amplitude (a.u.)

Normalized Amplitude (a.u.)

1

650

700

550

Wavelength (nm)

0.6

0.4

0.2

0

600

0.8

0

600

650

Wavelength (nm)

700

550

600

650

700

Wavelength (nm)(Figure(A4.!!Excitation!dependence!of!the!SRTG!response!of!mPlum!E16Q.!!!
Experimental#Temporal#Resolution#
The!length!of!excitation!pulse!and!excited!state!lifetime!of!our!excited!probe!
dictate!temporal!resolution!of!SRTG!data.!!For!RFPs,!lifetimes!range!from!~1V3!ns!
enabling!adequate!spectral!range!to!resolve!the!range!of!intramolecular!vibrations,!
solvent!motion,!and!sidechain!reorganization.!!However,!this!resolution!is!limited!in!
both!temporal!extremes.!!Concerning!fast!timescales,!experimental!precision!is!
limited!by!the!excitation!pulse!duration!(~20!fs)!in!principle.!!Examination!of!data!at!
early!delay!reveals!evidence!of!pulse!overlap!effects!extending!to!~150!fs!largely!
due!to!imperfect!compression.!!This!slightly!inhibits!resolution!of!the!fastest!
dynamics.!!Typical!pulse!overlap!distortions!at!early!delay!are!illustrated!in!Figure!
A5.!!In!the!case!of!mPlum,!the!interesting!dynamics!fall!in!the!picosecond!range!and!
their!measurement!is!uncompromised!by!these!spectral!artifacts.!!Conversely,!

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!
quantification!of!dynamics!on!long!timescales!is!limited!by!the!delay!stage!length!
(1.3!ns)!and!relaxation!of!excited!state!population.!!For!variants!with!short!lifetimes!
(~1!ns),!signal!intensity!begins!to!approach!background!scatter!and!steady!state!
fluorescence!around!700!ps!for!a!typical!sample.!!

Wavelength (nm)

700

650

600

550
0

0.1

0.2
0.3
Delay (ps)

0.4

0.5
(

Figure(A5.!!SRTG!data!for!mPlum!at!early!population!time.!!Pulse!overlap!effects!are!
evident!within!the!first!~150!fs!in!most!samples.!!!
Detection#
The!nonlinear!signal!field!can!be!detected!using!a!homodyne!or!heterodyne!
scheme!within!the!JILA!MONSTR!experimental!platform.!!The!latter!provides!a!
means!of!eliminating!contributions!from!excitation!scatter!and!steady!state!
emission,!however,!it!was!observed!that!the!spectral!envelope!of!the!signal!was!
extremely!sensitive!to!spectral!filtering!as!demonstrated!in!Figure!A6.!!For!this!
reason,!all!reported!SRTG!data!was!collected!utilizing!a!homodyne!detection!scheme.!!!

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!

Time cut 1
Time cut 2

1

Normalized Amplitude (a.u.)Time cut 3
0.8

0.6

0.4

0.2

0

0.25

0.3

0.35

0.4

0.45

0.5

0.55

Emission Time (ps)Time cut 1

Time cut 2

0.6

0.4

0.2

1

Normalized Amplitude (a.u.)

Normalized Amplitude (a.u.)

Normalized Amplitude (a.u.)

0.8

0.8

0.6

0.4

650

Wavelength (nm)

700

550

0.6

0.4

0

0

600

0.8

0.2

0.2

0

550

Time cut 3

1

1

600

650

Wavelength (nm)

700

550

600

650

700

Wavelength (nm)!
Figure(A6.!!Examples!of!heterodyneVdetected!SRTG!data!of!mPlum!with!various!
temporal!filters.!!Varying!the!temporal!window!yields!interference!within!the!
spectral!envelope!that!complicates!extraction!of!excited!state!dynamics.!!!
Survey#of#other#RFPs#
The!results!of!mPlum,!presented!in!Chapter!III,!motivated!a!survey!of!the!
solvation!dynamics!across!the!mFruit!family!and!related!RFPs.!!A!representative!
collection!of!data!for!several!species!is!presented!in!Figure!A7.!!It!was!discovered!
that!quantification!dynamics!in!these!species!was!difficult!for!a!few!reasons.!!The!
primary!obstacle!to!measurement!of!excited!state!solvation!was!lack!of!an!obvious!
SE!band!in!most!cases.!!Resolution!of!this!response!might!be!due!to!either!
inadequate!Stokes!shift!or!presence!of!an!overlapping!ESA!band!as!observed!in!
transient!absorption!measurements.!!Given!that!the!GSB!component!also!shows!
little!movement!over!the!experimental!time!window,!it!is!likely!that!chromophore!

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!
environment!is!rigid!in!these!species!compared!to!mPlum!and!their!Stokes!shift!may!
originate!in!some!other!way.!!This!further!implicates!the!importance!of!the!NV
acylimine!carbonylVposition!41!sidechain!interaction!as!a!primary!mechanism!of!
Stokes!shift!in!red!fluorescent!proteins.!

mApplemCherry

0.6

0.4

0.2

1

Normalized Amplitude (a.u.)

0.8

0.8

0.6

0.4

0.2

0

500

mOrange2

1

Normalized Amplitude (a.u.)

Normalized Amplitude (a.u.)

1

540

560

580

600

620

640

660

0.4

0

540

560

580

600

Wavelength (nm)

620

640

660

680

700

720

540

mStrawberrymNeptune

0.4

0.8

0.6

0.4

0

0

600

620

640

660

550

610

0.4

0

580

600

0.6

0.2

Wavelength (nm)

590

0.8

0.2

560

580

mKate

0.2

540

570

1

Normalized Amplitude (a.u.)

Normalized Amplitude (a.u.)

0.6

520

560

Wavelength (nm)

1

0.8

500

550

Wavelength (nm)1Normalized Amplitude (a.u.)

0.6

0.2

0

520

0.8

600

650

Wavelength (nm)Figure(A7.!!SRTG!survey!of!several!RFPs.!!
(
!
!

700

540

560

580

600

620

Wavelength (nm)

640

660

680

700

!