Telluride Science Research Center
2023Protein Dynamics Workshop
July 24-28, 2023
Arthur Palmer and Takahisa Yamato

Participants (19 lectures)
Participant

RegionEmailAndo, Nozomi

USAnozomi.ando@cornell.edu

Anfinrud, PhilipUSAanfinrud@nih.gov

Bruschweiler, RafaelUSA

bruschweiler.1@chemistry.ohio-state.edu

Fenimore, PaulUS

paulf@lanl.gov

Hayashi, ShigehikoAsiahayashig@kuchem.kyoto-u.ac.jp

Iino, Ryota

Asia

iino@ims.ac.jp

Kitao, Akio

Asiaakitao@bio.titech.ac.jp

Massi, Francesca

USFrancesca.Massi@umassmed.edu

Mori, Toshifumi

Asia

toshi_mori@cm.kyushu-u.ac.jp

Nishizaka, Takayuki

Asiatakayuki.nishizaka@gakushuin.ac.jp

Oroguchi, TomotakaAsia

oroguchi@phys.keio.ac.jp

Palmer, ArthurUS

agp6@cumc.columbia.edu

Schlichting, Ilme

Europeilme.schlichting@mpimf-heidelberg.mpg.de

Straub, JohnUS

straub@bu.edu

Thielges, MeganUSthielges@indiana.edu

Tobias, Douglas

USdouglas.tobias@uci.edu

van Thor, Jasper

Europej.vanthor@imperial.ac.uk

Weik, Martin

Europemartin.weik@ibs.fr

Yamato, Takahisa

Asia

yamato@nagoya-u.jp

Workshop Schedule(All sessions are at the Intermediate School, 725 West Colorado Ave.)
Sunday, July 23, 5:00-6:30 pm
All-Telluride Science Meet and Greet
Location: Oak – 250 W San Juan Avenue (at the base of the gondola)This is a good chance to meet up with fellow participants prior to your meeting. A staff
member will be on hand to welcome you and distribute badges.
Monday, July 24
7:30-8:30

Breakfast at TSRC (Intermediate School)

8:30-9:00

Opening comments

Session 1: John Straub, Moderator
9:00-9:50
Takahisa Yamato
9:50-10:20
Break
10:20-11:10
Rafael Brüschweiler
11:10-12:00

Nozomi Ando

12:00-14:00

Energy and heat flow in proteins
Quantitative prediction of ensemble
dynamics, shapes and contact propensities
of intrinsically disordered proteins
Correlated atomic displacements in proteins

Lunch (not provided)

Session 2: Toshifumi Mori, Moderator
14:00-14:50
Akio Kitao
14:50-15:40

Douglas Tobias

15:40-16:10
16:10-17:00

BreakJohn Straub

Rotational mechanisms of bacterial flagellum
and roles of water
Voltage-dependent structural dynamics ofthe Hv1 Proton Channel
Understanding amyloid protein aggregationfrom the perspective of the monomer
conformational ensemble

Tuesday, July 25
7:30-9:00

Breakfast at TSRC (Intermediate School)

9:00-14:00

Free time

Session 3: Ilme Schlichting, Moderator
14:00-14:50
Megan Thielges
14:50-15:40

Philip Anfinrud

Site-selective infrared spectroscopy toinvestigate protein conformations anddynamics
Probing biomolecule structure and dynamics
as a function of temperature via timeresolved small- and wide-angle X-ray

scattering
15:40-16:10
15:10-17:00

BreakTomotaka Oroguchi

Optimization of MD-derived conformational
ensemble in information content space andits application to experimental X-ray solution
scattering data

18:30-19:20
Telluride Science Town Talk
THE TRUE COLORS OF CANCER: Imaging New Biomarkers with Light, not Labels
Stephen Boppart, M.D, PH.D
Professor and Grainger Distinguished Chair in Engineering
University of Illinois Urbana-Champaign
Location: Telluride Conference Center in Mountain Village
Cash Bar, Doors Open at 18:00.
Wednesday, July 26
7:30-9:00

Breakfast at TSRC (Intermediate School)

Session 4: Megan Thielges, Moderator
9:00-9:50
Martin Weik

Photoswitchable fluorescent proteins: What
we have learnt through time-resolved serial
crystallography
Coherence Structure from non-linear opticalcrystallography and Coherent control ofultrafast structural dynamics withfemtosecond X-ray crystallography

9:50-10:40

Jasper van Thor

10:40-11:10
11:10-12:00

BreakPaul Fenimore

12:00-14:00

Lunch break (not provided)

TBA

Session 5: Jasper van Thor, Moderator
14:00-14:50

Ilme Schlichting

14:50-15:40

Takayuki Nishizaka

15:40-16:10
16:10-17:00

Break
Ryota Iino

Mechanism and dynamics of fatty acidphotodecarboxylase
Insights into the mechanism of ATP-driven
rotary motors from direct torquemeasurement
Single-molecule analysis and engineering ofmolecular motor proteins

17:30-19:30
All Telluride Science Picnic
Free BBQ, Beer, Wine and Non-Alcoholic Beverages. Friends and Family are invited
free of charge.
Location: Tent behind the Intermediate School.

Thursday, July 27
7:30-9:00

Breakfast at TSRC (Intermediate School)

9:00-14:00

Free time

Session 6: Takahisa Yamato, Moderator
14:00-14:50
Toshifumi Mori

Dissecting the heterogeneous dynamics ofproteins during folding and catalysis
The CCCH-type zinc finger proteins:functional insights from an ancient family of
post-transcriptional regulators

14:50-15:40

Francesca Massi

15:40-16:10
16:10-17:00

Break
Shigehiko Hayashi

17:00-17:50

Arthur Palmer

17:50-18:20

Summing up, selection of next organizing committee

Atomistically deciphering functional activation
processes of transporter proteins withmolecular simulations
Conformational dynamics in molecular
recognition

Friday, July 28
7:30-9:00

Breakfast at TSRC (Intermediate School)

Workshop Abstracts
Correlated Atomic Displacements in Proteins
Nozomi Ando
Chemistry & Chemical Biology
Cornell University
Correlated motions in proteins arising from the collective movements of residues
have long been proposed to be fundamentally important to key properties of proteins, from
catalysis and allostery to evolvability. Recent breakthroughs in structural biology have madeit possible to capture proteins undergoing complex conformational changes, yet the subtle
and intrinsic fluctuations within a single conformation remain one of the least understood
facets of protein structure. For many decades, the analysis of diffuse X-ray scattering held
the promise of animating crystal structures with such correlated motions. With recent
advances in both X-ray detectors, this long-held promise can now be met. In this talk, I’ll
describe our recent work in interpreting this signal and discuss how it can be used to probe
structural fluctuations that give rise to fundamental protein properties.
Probing Biomolecule Structure and Dynamics as a Function of Temperature via
Time-resolved Small- and Wide-Angle X-ray Scattering
Philip Anfinrud, Hyun Sun Cho, Friedrich Schotte, and Eli WorthNational Institutes of Health, NIDDK, Laboratory of Chemical Physics
Life flourishes over a broad range of temperatures spanning from around -2 Celsius
for arctic fish to 122 Celsius for hyperthermophillic Methanopyrus kandleri. To understandhow biomolecules function over such a broad temperature range, it is crucial to develop
methods capable of probing temperature-dependent structure and structure dynamics at
extreme temperatures. To that end, we continue to refine x-ray scattering methods recently
developed on the BioCARS beamline at the Advanced Photon Source to probe biomolecule
structural dynamics in solution. For example, Small Angle X-ray Scattering (SAXS) data canunveil biomolecule size and shape as well as biomolecule-biomolecule interactions.
Moreover, Wide Angle X-ray scattering (WAXS) data can be used to characterize sample
temperature and secondary structure. We have acquired temperature-dependent and timeresolved x-ray scattering data from proteins of various sizes as well as from various RNA
constructs including hairpins and dumbbells. The structural details extracted from these
SAXS/WAXS studies extend beyond that accessible with other biophysical techniques, andprovide a wealth of information that can help unveil mechanistic details of protein
folding/unfolding and function.

Quantitative Prediction of Ensemble Dynamics, Shapes and Contact {ropensities ofIntrinsically Disordered Proteins
Rafael Brüschweiler
Departments of Chemistry & Biochemistry and Biological Chemistry & Pharmacology
The Ohio State University
Intrinsically disordered proteins (IDPs) are highly dynamic systems that play animportant role in cell signaling processes and their misfunction often causes humandisease. Proper understanding of IDP function not only requires the realistic
characterization of their three-dimensional conformational ensembles at atomic-level
resolution but also of the time scales of interconversion between their conformational
substates. Large sets of experimental data are often used in combination with molecularmodeling to restrain or bias models to improve agreement with experiment. It is shown for
the N-terminal transactivation domain of p53 (p53TAD) and Pup, which are two IDPs thatfold upon binding to their targets, how the latest advancements in molecular dynamics (MD)simulations methodology produces native conformational ensembles by combining replica
exchange with series of microsecond MD simulations. They closely reproduce experimental
data at the global conformational ensemble level, in terms of the distribution properties of
the radius of gyration tensor, and at the local level, in terms of NMR properties including 15N
spin relaxation, without the need for reweighting. The IDP ensembles were analyzed bygraph theory to identify dominant inter-residue contact clusters and characteristic aminoacid contact propensities. These findings indicate that modern MD force fields with residuespecific backbone potentials can produce highly realistic IDP ensembles sampling ahierarchy of nano- and picosecond time scales providing new insights into their biological
function.
Atomistically Deciphering Functional Activation Processes of Transporter Proteins
with Molecular Simulations
Shigehiko Hayashi
Graduate School of Science
Kyoto University, Japan
Activation processes of protein functions are often fulfilled by dynamic and global
molecular conformational changes of complex protein systems which correlate with local
molecular events at chemically active sites. Hence the multi-scale functional coupling of
local chemical events with protein global molecular dynamics needs to be revealed forunderstanding of molecular nature of protein functions.
In this talk, I will present our recent studies on activation processes of photo-sensitive iontransporters by a hybrid QM/MM free energy geometry optimization technique, which allows
one to optimize electronic wave function and molecular geometry of the chemically active
sites at the ab initio quantum chemistry level of theory on a free energy surface constructedwith statistically extensive conformational ensemble of the protein environment obtained bylong-time MD simulations [1].
We succeeded in theoretically predicting photo-activated structural changes of aphoto-sensitive cation membrane channel, C1C2 channelrhodopsin, which remarkably
differ from previously known conformational changes in other homologous proteins [2] andwere verified by a recent time resolved serial femtosecond X-ray crystallographymeasurement [3]. We also simulated photo-activation processes of anion membranetransporters and revealed significant conformational changes of the channels. We firstexamined protonation states of carboxy groups in the channel which strongly affecttransport properties of ions. Through excitation energy calculations of various mutants, we
determined the protonation states which are consistent with experimental evidence. We
also succeeded in modeling a photo-activated channel opening structure by the hybrid
QM/MM free energy optimization with a long-time MD simulation. Free energy profiles of
the ion transport in the channel opening state were then obtained by umbrella sampling MD
simulations, revealing atomistic dynamics and energetics of the ion transport process.
I will also present an atomistic molecular dynamics (MD) study of alternating accessof mitochondria ADP/ATP transporter with a linear response path following method [4,5]which is a biasing MD technique accelerating global protein conformational changes
coupled to local ligand binding events. The proposed structural model of the alternating
access state has been shown to successfully predict at atomic resolution an X-ray
crystallographic one reported later.
[1] Hayashi et al., Annu. Rev. Phys. Chem. 68, 135 (2017). [2] Cheng et al., Biophys. J.,
115, 1281 (2018). [3] Oda et al., eLife, 10, e62389 (2021). [4] Tamura and Hayashi, J.
Chem. Theory and Comput., 11, 2900-2917 (2015). [5] Tamura and Hayashi, PLOS ONE,12, e0181489 (2017)
Single-molecule Analysis and Engineering of Molecular Motor Proteins
Ryota Iino
Institute for Molecular Science
National Institutes of Natural Sciences, Japan
Molecular motors, an important class of molecular machines, harness various energy
sources to move unidirectionally [1]. The operational principles of molecular motors are
distinct from those of man-made macroscopic motors, because they have nanoscaledimensions and generally work in a solution environment where viscosity is dominant.
Under these low Reynolds number, overdamped conditions, they cannot rely on inertia tosustain motion. Furthermore, they are continually agitated by random Brownian motion,
which provides both challenges and opportunities for the unidirectional motion.
To understand the operational principles of molecular motor proteins, we aredeveloping single-molecule imaging techniques which enable visualization of their fast anddynamic motions with microsecond time resolution and angstrom localization precision [2,3]. Furthermore, to understand their design principles and to outperform natural ones, weare engineering non-natural molecular motor proteins by using saturation mutagenesis [4],hybrid/chimera generation [5], and computational design [6]. In this seminar, I will overview
our approaches by introducing our results on rotary and linear motors such as V-ATPase [7,8], processive chitinase [9], kinesin [10], and dynein [11].
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Ando J, et al., Biophys J, 115, 2413-2427 (2018)
Ando J, et al., ACS Photonics, 6, 2870-2883 (2019)
Visootsat A, et al., ACS Omega, 5, 26807–26816 (2020)
Baba M, et al., PNAS, 113, 11214-11219 (2016)
Kosugi T, et al., Nat Chem, in press
Iida T, et al., J Biol Chem, 294, 17017-17030 (2019)
Otomo A, et al., Proc Natl Acad Sci USA 119, e2210204119 (2022)
Nakamura A, et al., Nat Commun, 9, 3814 (2018)

10. Isojima H, et al., Nat Chem Biol, 12, 290-297 (2016)
11. Ando J, et al., Sci Rep, 10, Article number: 1080 (2020)
Rotational mechanisms of bacterial flagellum and roles of water
Akio Kitao
School of Life Science and Technology
Tokyo Institute of Technology, Japan
Many pathogenic bacteria, such as Escherichia coli and Salmonella, use bacterial
flagella to propel. Bacterial flagellum is a large molecular machine, whose length can be 10
micrometers long, and it is an assembly of ~30,000 molecules of more than 30 different
proteins. For cell motility, bacterial flagellum employs a transport apparatus to construct a
giant molecular assembly, converts ionic gradient energy into rotational force, and transmits
the rotational force via a flagellar rod and hook (universal joint) to rotate the flagellar
filament (screw). The rotation is controlled by chemotaxis signals, and cell motility isregulated by changing the helical nature of the flagellar filament.
We investigated the mechanisms by which the flagellar rod transmits torquesupported by the flagellar bushing, allowing rotation at low energy cost with large-scale
molecular dynamics simulations. 40 nanosecond molecular dynamics simulations were
performed from the initial states of 140 different rotation angles of rod-hook complex. The
free-energy landscape of the rotation and the time required for the rotation were calculatedby the Markov state model. As a result, it was found that the rod-hook complex can rotate at~ 1900 Hz, which is consistent with the time obtained experimentally so far. We are
currently analyzing the interactions between the rod-hook complex and the LP ring
mediated by water molecules in between, which enables rotations at low energy cost while
maintaining the structure of the large flagellar complex.
The CCCH-type Zinc Finger Proteins: Functional Insights from an Ancient Family of
Post-transcriptional Regulators
Francesca Massi
Biochemistry and Molecular Biotechnology
UMass Chan Medical School
CCCH-type tandem zinc finger (TZF) domains are found in many proteins that are
involved in transcriptional regulation and splicing. We have discovered that these TZF
domains have evolved to have different propensities toward intrinsic disorder and recognize
diverse RNA sequences. We investigate how the structure and dynamics of these TZF
domains impact differential RNA recognition, localization, and turnover and allow for
regulation of the protein activity.

Dissecting the Heterogeneous Dynamics of Proteins During Folding and
Catalysis
Toshifumi Mori
Institute for Materials Chemistry and Engineering
Kyushu University, Japan
While the structures of proteins have been critical for understanding functions, theimportance of dynamics has become evident over the last decades. Molecular dynamics
simulation is a powerful tool in studying the structures and functions of biomolecules inmolecular detail, but most computational studies have focused on the static (free energy)
and kinetic perspectives while often omitting the dynamic aspect. Furthermore, how protein
dynamics participate in enzyme catalysis have been highly debated [1,2].
To decipher the role of dynamics in protein function, we have been developing
theoretical approaches and studying the dynamic aspect of proteins using molecular
dynamics simulations. Here I discuss the dynamics and its role behind protein folding [3,4]and enzyme catalysis [5,6,7]. The analyses of protein folding trajectories shows that protein
folding occurs via multiple pathways, and furthermore, the transitions occur in a highly
heterogeneous manner. The studies of the enzymatic reaction catalyzed by the peptidylprolyl isomerase Pin1, show that protein conformational transition precedes the reaction
event, i.e., isomerization [5]. The lag in protein and ligand dynamics in enzyme catalysis
occurs due to the separation of the temporal scales. Thus, the distinctive protein
conformation necessary for the reaction to proceed is only set up as a conformational
excited state. Further analysis of the slow dynamics of Pin1 indicates that changes in theequilibrium distribution of the Pin1’s conformational ensembles before and after the reaction
results in a seeming coupling of protein dynamics and chemical reactions during catalysis[7,8].
1. S. D. Schwartz, V. L. Schramm, Nat. Chem. Biol. 5, 551–558 (2009)
2. A. Warshel, A. R. P. Bora, J. Chem. Phys. 144, 180901 (2016).
3. T. Mori, S. Saito, J. Chem. Phys. 142, 135101 (2015)
4. T. Mori, S. Saito, J. Phys. Chem. B 120, 11683 (2016)
5. T. Mori, S. Saito, J. Phys. Chem. Lett. 10, 474–480 (2019).
6. T. Mori, S. Saito, J. Chem. Theory Comput. 16, 3396–3407 (2020).
7. T. Mori, S. Saito, J. Phys. Chem. B 126, 5185–5193 (2022)
8. W. Labeikovsky et al., J. Mol. Biol. 367, 1370–1381 (2007).
Insights into the Mechanism of ATP-driven Rotary Motors from Direct TorqueMeasurement
Takayuki Nishizaka
Department of Physics, Gakushuin University, Japan
Motor proteins are molecular machines that convert chemical energy into mechanicalwork. In addition to existing studies performed on the linear motors found in eukaryotic
cells, researchers in biophysics have also focused on rotary motors such as bacterial
flagellar motor (BFM) and F1-ATPase. The research group including Nishizaka contributed
to correlate all chemical states to specific mechanical events by visualizing single chemical
reactions under the advanced optical microscope [1-3]. From the structural point of view,
conformational changes of β-subunit [4,5] and behaviours of the γ-shaft [6-8] were also
addressed through a series of microscopy techniques at the single molecular level. Recent

studies showed that there exists another ATP-driven protein motor in life: the rotary
machinery that rotates archaeal flagella (archaella). None of the archaellar motility structureis homologous to any BMF proteins. Rotation speed, stepwise movement, and variabledirectionality of the motor of Halobacterium salinarum were described in previous studies[9]. We further presented the experimental work discerning the molecular mechanism
underlying how the archaellar motor protein FlaI drives rotation by generation of motor
torque [10]. In combination, those studies found that rotation slows as the viscous drag ofmarkers increases, but torque remains constant at 160 pN·nm independent of rotation
speed. Unexpectedly, the estimated work done in a single rotation is twice the expectedenergy that would come from hydrolysis of six ATP molecules in the FlaI hexamer. To
reconcile the apparent contradiction, a new and general model for the mechanism of ATPdriven rotary motors [11] will be discussed.
1.
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Nishizaka, T., K. Oiwa, H. Noji, S. Kimura, E. Muneyuki, M. Yoshida, K. Kinosita, Jr.,Nat Struct Mol Biol, 2004. 11(2): p. 142-8.
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Kinosita, Jr., Cell, 2007. 130(2): p. 309-21.
Adachi, K., K. Oiwa, M. Yoshida, T. Nishizaka, K. Kinosita, Jr., Nat Commun, 2012.
3: p. 1022.
Masaike, T., F. Koyama-Horibe, K. Oiwa, M. Yoshida, T. Nishizaka, Nat Struct Mol
Biol, 2008. 15(12): p. 1326-33.
Sugawa, M., K. Okazaki, M. Kobayashi, T. Matsui, G. Hummer, T. Masaike, T.
Nishizaka, Proc Natl Acad Sci USA, 2016. 113(21): p. E2916-24.
Sugawa, M., K.A. Okada, T. Masaike, T. Nishizaka, Biophys J, 2011. 101(9): p.
2201-6.
Naito, T.M., T. Masaike, D. Nakane, M. Sugawa, K.A. Okada, T. Nishizaka, Sci Rep,
2019. 9(1): p. 7451.
Hasimoto, Y., M. Sugawa, Y. Nishiguchi, F. Aeba, A. Tagawa, K. Suga, N. Tanaka,
H. Ueno, H. Yamashita, R. Yokota, T. Masaike, T. Nishizaka, Biophys J, 2023.
122(3): p. 554-564.
Kinosita, Y., N. Uchida, D. Nakane, T. Nishizaka, Nat Microbiol, 2016. 1(11): p.
16148.
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Nishizaka, T., T. Masaike, D. Nakane, Biophys Rev, 2019. 11(4): p. 653-657.

Optimization of MD-derived Conformational Ensemble in Information Content Spaceand its Application to Experimental X-ray Solution Scattering Data
Tomotaka Oroguchi
Department of Physics, Keio University, Japan
The information on protein conformational ensemble is essential for understanding
the mechanisms of protein function. MD simulation is a powerful technique to acquire suchinformation. However it is often difficult for MD simulations to provide a conformational
ensemble that can reproduce experimental data, due to the low reliability of force fields andlow efficiency of conformational sampling. Therefore, optimization of the MD-derived
conformational ensemble is required to achieve consistency with experimental data. In thisstudy, we propose an optimization method using the information content space. In themethod, the ensemble model as a probability density distribution is first projected on theinformation content space. Then the model is iteratively moved toward the optimized modelin this space according to a newly introduced Lagrangian, which was constructed based on
the consistency between the model and experimental data. To evaluate the validity of thealgorithm, simulations of the optimization process assuming X-ray solution scattering(SAXS) as an experiment were performed using test conformational ensembles generated
via coarse-grained MD. These simulations demonstrated the possibility of obtaining true
conformational ensembles using our method with SAXS data. We also applied the method
to experimental SAXS data of glucosamine kinase, and revealed the changes inconformational ensemble on substrate binding.
Conformational dynamics in molecular recognition
Arthur Palmer
Biochemistry and Molecular Biophysics
Columbia University
Recognition of substrates and other ligands is an essential part of the mechanismsof biological function of proteins, in processes including catalysis, regulation, and signaltransduction. Interactions between proteins and other molecules frequently require
conformational changes in either protein or target or both, or more precisely a shift in theconformational ensemble between the apo and holo states of the system. NMR
spectroscopy and molecular dynamics (MD) simulations are complementary experimental
and computational methods, respectively, for describing conformational dynamics ofproteins with atomistic and temporal resolution. The latest results in on-going studies of theribonuclease H superfamily and the GCN4 transcription factor will be presented as model
systems for elucidating linkages between conformational dynamics and molecular
recognition by proteins.
Mechanism and Dynamics of Fatty Acid Photodecarboxylase
Ilme Schlichting
Max Planck Institute for Medical Research, Germany
Light is important for organisms from all domains of life, serving as an energy
resource or carrier of information initiating intra- or intercellular signaling. Photosensitive
proteins, endowed with a light-absorbing chromophore, enable this. Detailed insights,
including the initial events on the ultrafast time scale, can be obtained by various forms ofspectroscopy and computation. However, direct structural information necessary tounderstand the underlying molecular mechanisms has been inaccessible until recently. The
unique properties of X-ray free electron lasers open the sub-ps time domain for timeresolved crystallography using small crystals that can be efficiently photolyzed, thus
providing access to the long sought-after excited state and intermediate structures.
Photodecarboxylation is a well-established reaction in chemistry; however, no photoenzymatic equivalent was known until the discovery of Fatty Acid Photodecarboxylase(FAP), a flavin containing photoenzyme [1]. The enzymatic mechanism was investigated indetail by a large interdisciplinary consortium [2]: decarboxylation occurs directly upon
reduction of the photo-excited flavin by the fatty acid substrate. Along with flavin reoxidationby the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide
unexpectedly transforms in 100 ns into another species, assigned to be bicarbonate basedon IR-spectroscopy performed on cryogenic samples. Despite a great deal of insight intothe catalytic mechanism and the role of two strictly conserved residues for substratestabilization and functional charge transfer [2], a number of questions remain. To addressthese, including the nature of the transiently generated CO2-derivative we performed afollow-up time-resolved serial femtosecond crystallography experiment at SwissFEL
spanning time-delays from ns to ms after photoexcitation. The results will be discussed.
[1] Sorigué et al An algal photoenzyme converts fatty acids to hydrocarbons, Science 357:
903-907 (2017)[2] Sorigué et al Mechanism and dynamics of fatty acid photodecarboxylase, Science 372,
eabd5687 (2021)

Understanding Amyloid Protein Aggregation from the Perspective of the Monomer
Conformational Ensemble
John Straub
Boston University
A crucial step in the aggregation of amyloidogenic sequences is the transition from a
relatively disordered state to assembly-competent structures referred to as N* states. The
assembly-competent N* states share key structural characteristics with the protein
monomer in the fibril environment as observed in experiments. Using a coarse-grainedmodel for intrinsically disordered proteins (IDPs), we demonstrate that the N* states exist as
rare excited states on the monomer free energy landscape. By relating the populations of
the N* states to fibril formation time scales, using an empirical relation derived from latticesimulations, we quantitatively correlate the probability of observing the N* states with therelative aggregation propensities. Using kinetic transition networks, we observe that the N*
states are obligatory along the favored assembly routes. I will discuss recent applicationsof N* theory to explore the origins of polymorphism in fibrils composed of amyloid-β (Aβ)
peptide and fibrils composed of fused-in-sarcoma (FUS) peptide.
Site-selective Infrared Spectroscopy to Investigate Protein Conformations andDynamics
Megan Thielges
Department of Chemistry
Indiana University
To fully uncover the role of protein dynamics in function, experimental approaches
must contend with both the complex spatial heterogeneity of proteins and the rapid
interconversion of potentially important states. To address these challenges, our groupcombines the inherent temporal resolution of linear and two-dimensional infrared
spectroscopy with the spatial resolution afforded by site-selective incorporation ofvibrational reporter groups. This enables detection of individual vibrations to probe specificlocations and capture of even rapid dynamics throughout proteins. I will share how we apply
this approach to investigate the functional role of dynamics in protein molecular recognitionand catalysis.

Voltage-dependent structural dynamics of the Hv1 Proton Channel
Andrew Geragotelis,1 Mona L. Wood,1 Hendrik Goeddeke,1 Quinn Nguyen,1 Eric K. Wong,1
J. Alfredo Freites,1 Liang Hong,2 Parker D. Webster,2 Francesco Tombola,2and Douglas J. Tobias1
Departments of Chemistry1 and Physiology and Biophysics2
University of California, Irvine
The voltage-gated Hv1 proton channel is a ubiquitous membrane protein that plays
roles in a variety of cellular processes, including proton extrusion, pH regulation, productionof reactive oxygen species, proliferation of cancer cells, and increased brain damage during
ischemic stroke. Like voltage-gated metal ion channels, Hv1 contains a voltage-sensing
domain (VSD), but it lacks a separate ion-conducting pore domain. The functional unit of
Hv1 is a dimer in which the monomers are connected by a coiled-coil domain (CCD).
Proton conduction occurs within each of the two VSDs and monomeric constructs lacking
the CCD also conduct protons. The Hv1 VSDs open and close in response to changes inthe transmembrane (TM) electric potential. In order to elucidate the voltage-dependent
structures and the transition between the closed and open states of Hv1, we carried out
multi-microsecond molecular dynamics (MD) simulations of the human Hv1 (hHv1)
monomer under physiologically relevant applied electric fields in an explicit membrane
environment. The crystal structure of the mouse Hv1 chimera (mHv1cc), which shares asequence identity of 65% with hHv1 in the TM regions, was used as a template forhomology modelling of hHv1. In simulations of the unpolarized and hyperpolarized/restingstates, the hHv1 monomer contains a complex salt-bridge network and extensive internal
hydration. In response to a depolarizing membrane potential the voltage-sensing S4 helix
undergoes a large outward displacement, leading to changes in the VSD internal salt-bridge
network, resulting in a reshaping of the proton permeation pathway and a significant
increase in the hydrogen-bond connectivity throughout the channel. The total gating charge
displacement associated with this transition is consistent with experimental estimates.
Docking calculations confirm the proposed mechanism for the inhibitory action of the first
known Hv1 blocker, 2-guanidinobenzimidazole, derived from electrophysiology
measurements and mutagenesis. Our depolarized structural model was also validated withthe formation of a metal bridge between residues located in the VSD. Taken together, ourresults suggest that the structures sampled in our MD simulations are representative of theclosed and open states of the Hv1 proton channel. Preliminary results from our on-going
efforts to model the Hv1 dimer will also be presented.

Coherence Structure from Non-linear Optical Crystallography and Coherent
Control of Ultrafast Structural Dynamics with Femtosecond X-ray Crystallography
Jasper van Thor
Imperial College, London
Non-linear spectroscopy measures the electronic and nuclear configuration but lacks
molecular structure information. X-ray crystallography determines molecular structure andcan also measure its dynamics, but lacks the electronic and nuclear configuration
information. Therefore crystallography can learn from spectroscopy and spectroscopy canlearn from crystallography.
We have demonstrated a coherent control experiment of vibrational coherence withultrafast X-ray crystallographic observation [1]. The photoisomerisation reaction of afluorescent protein chromophore includes ultrafast barrier crossings and the avoidance ofinternal conversion processes. The relationship between experimentally observed ultrafast
nuclear motions and the biological reaction coordinate is not known. High resolution pumpprobe X-ray crystallography measurements reveal complex sub-Ångström ultrafast motional
dynamics and hydrogen bonding rearrangements in the active site. However, wedemonstrate that the measured motions are not part of the reaction coordinates but insteadarise from impulsively driven coherent vibrational processes of the electronic ground state.
We apply the analysis of impulsive stimulated Raman spectroscopy and coherent control
methodology to the real-space observation of low frequency vibrational dynamics anddisplacements, that we have measured by ultrafast X-ray crystallography. Using the‘Tannor-Rice’ coherent control method it is shown that ultrafast motions can be strongly
amplified. This demonstrates that the conventional pump-probe measurements, without
application of optical control, are dominated by electronic ground state displacements thatare unrelated to the reactive photoisomerisation coordinate. We present the first applicationof coherent control using X-ray crystallography in combination with coherence theory thathas application to the real space wavepacket observation. This allows the unambiguous
assignment of the structural changes observed in ultrafast X-ray crystallography.
Coherence analysis of non-linear spectroscopy can add structural information in the
x-ray crystallographic frame if measurements are made on oriented single crystals [1-5].
The third order response of crystals is generally different from measurements of isotropic
solutions because each coherence path that contributes to the measured field scales to theensemble average of the four-point correlation functions of the four field-dipole interactions
involved in the respective Feynman paths. The application of a symmetry operator to thepoint correlation function allows the explicit calculation of the structural dependence of allcontributing Feynman diagrams and the total signal field [5]. This level of structural analysis
is possible for non-linear optical crystallography but is also has applications for time
resolved serial femtosecond x-ray crystallography.
[1]. Christopher D.M. Hutchison, James M. Baxter, Ann Fitzpatrick, Gabriel Dorlhiac,
Alisia Fadini, Samuel Perrett, Karim Maghlaoui, Salomé Bodet Lefèvre, Violeta CordonPreciado, Josie L. Ferreira, Volha Chukhutsina, Douglas Garratt, Jonathan Barnard,
Gediminas Galinis, Flo Glencross, Rhodri M. Morgan, Sian Stockton, Ben Taylor, Letong
Yuan, Matthew, G. Romei, Chi-Yun Lin, Jon P. Marangos, Marius Schmidt, Viktoria
Chatrchyan, Tiago Buckup, Dmitriy Morozov, Jaehyun Park, Sehan Park, Intae Eom,
Minseok Kim, Dogeun Jang, Hyeongi Choi, HyoJung Hyun, Gisu Park, Eriko Nango, Rie
Tanaka, Shigeki Owada, Kensuke Tono, Daniel P. DePonte, Sergio Carbajo, Matt
Seaberg, Andy Aquila, Sebastien Boutet, Anton Barty, So Iwata, Steven G. Boxer, Gerrit
Groenhof, Jasper J. van Thor*. (2023) Optical control of ultrafast structural dynamics in afluorescent protein. Nature Chemistry, In press[2]. Hutchison CDM, Fadini A, van Thor JJ* (2022) Linear and Non-Linear
Population Retrieval with Femtosecond Optical Pumping of Molecular Crystals for the
Generalised Uniaxial and Biaxial Systems, Applied Sciences Vol: 12[3]. Bressan G, van Thor JJ*, (2021) Theory of two-dimensional spectroscopy withintense laser fields, The Journal of Chemical Physics, Vol: 154, Pages: 1-10[4]. van Thor JJ* (2019) Advances and opportunities in ultrafast X-ray
crystallography and ultrafast structural optical crystallography of nuclear and electronic
protein dynamics, Structural Dynamics, Vol: 6, 050901[5] van Thor JJ* (2019) Coherent two-dimensional electronic and infrared
crystallography, The Journal of Chemical Physics, Vol: 150, 124113

Photoswitchable Fluorescent Proteins: What We Have Learnt Through Timeresolved Serial Crystallography
Martin Weik,
Institut de Biologie Structurale, France
Reversibly photoswitchable fluorescent proteins (RSFP) are essential tools in
advanced fluorescence nanoscopy of live cells (such as PALM and RESOLFT). They canbe repeatedly toggled back and forth between a fluorescent (on) and a non-fluorescent(off) state by irradiation with light at two different wavelengths.
We combined time-resolved serial femtosecond crystallography (TR-SFX), ultrafast
absorption spectroscopy in solution and in microcrystals and simulation methods to study
off-to-on photoswitching mechanisms in the RSFP rsEGFP2 on the nano- to picosecond
time scale. Off-to-on photoswitching proceeds via excited-state isomerisation, as
evidenced by capturing an intermediate with a fully twisted chromophore 1 ps after photon
absorption [1], followed by ground-state chromophore deprotonation [2]. Through the TRSFX studies we discovered that the cis chromophore of rsEGFP2 isomerizes to two
distinct off-state conformations, trans1 and trans2, located on either side of the V151 side
chain that was shown to transiently retract on the picosecond time scale during off-to-on
photoswitching [1]. Reducing or enlarging the side chain at this position (V151A and V151L
variants) leads to single off-state conformations (trans1 and trans2, respectively [3]). The
V151A variant was shown to exhibit higher photoswitching contrast, both in vivo and invitro, a property of prime interest for RESOLFT nanoscopy [3]. Our studies shed light on
the structural dynamics involved in off-to-on photoswitching and suggests a means torationally improve fluorescent proteins for nanoscopy applications. Currently, we are
studying the influence of the pump-laser fluence on structural changes seen in rsEGFP2, amuch-debated issue in the TR-SFX field.
The work presented involves a multi-national consortium composed by the authorsof references [1 – 3].
[1] Coquelle, N., M. Sliwa, J. Woodhouse, G. Schirò, V. Adam, A. Aquila, T. R. M.
Barends, S. Boutet, M. Byrdin, S. Carbajo, E. De la Mora, R. B. Doak, M. Feliks, F.
Fieschi, L. Foucar, V. Guillon, M. Hilpert, M. S. Hunter, S. Jakobs, J. E. Koglin, G.
Kovacsova, T. J. Lane, B. Lévy, M. Liang, K. Nass, J. Ridard, J. S. Robinson, C. M.
Roome, C. Ruckebusch, M. Seaberg, M. Thepaut, M. Cammarata, I. Demachy, M. Field,
R. L. Shoeman, D. Bourgeois, J.-P. Colletier, I. Schlichting and M. Weik (2018).
"Chromophore twisting in the excited state of a photoswitchable fluorescent protein
captured by time-resolved serial femtosecond crystallography." Nature Chemistry 10: 3137.
[2] Woodhouse, J., G. Nass Kovacs, N. Coquelle, L. M. Uriarte, V. Adam, T. R. M.
Barends, M. Byrdin, E. de la Mora, R. Bruce Doak, M. Feliks, M. Field, F. Fieschi, V.
Guillon, S. Jakobs, Y. Joti, P. Macheboeuf, K. Motomura, K. Nass, S. Owada, C. M.
Roome, C. Ruckebusch, G. Schirò, R. L. Shoeman, M. Thepaut, T. Togashi, K. Tono, M.
Yabashi, M. Cammarata, L. Foucar, D. Bourgeois, M. Sliwa, J.-P. Colletier, I. Schlichting
and M. Weik (2020). "Photoswitching mechanism of a fluorescent protein revealed by timeresolved crystallography and transient absorption spectroscopy." Nature Communications
11(1): 741.
[3] Adam, V., K. Hadjidemetriou, N. Jensen, R. L. Shoeman, J. Woodhouse, A. Aquila, A.
S. Banneville, T. R. M. Barends, V. Bezchastnov, S. Boutet, M. Byrdin, M. Cammarata, S.

Carbajo, N. Eleni Christou, N. Coquelle, E. De la Mora, M. El Khatib, T. Moreno Chicano,
R. Bruce Doak, F. Fieschi, L. Foucar, O. Glushonkov, A. Gorel, M. L. Grunbein, M. Hilpert,
M. Hunter, M. Kloos, J. E. Koglin, T. J. Lane, M. Liang, A. Mantovanelli, K. Nass, G. Nass
Kovacs, S. Owada, C. M. Roome, G. Schiro, M. Seaberg, M. Stricker, M. Thepaut, K.
Tono, K. Ueda, L. M. Uriarte, D. You, N. Zala, T. Domratcheva, S. Jakobs, M. Sliwa, I.
Schlichting, J. P. Colletier, D. Bourgeois and M. Weik (2022). "Rational Control of Off-State
Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast
Enhancement." Chemphyschem 23(19): e202200192.
Energy and Heat Flow in Proteins
Takahisa Yamato
Graduate School of Science
Nagoya University, Japan
Although thermal energy transport is among the essential biophysical properties ofproteins, its relationship with protein structure, dynamics, and functions is still elusive [1].
Within thermally fluctuating protein molecules, tightly packed amino acid residues interact
with each other through heat and energy exchanges [2]. The structures of folded proteins
are highly inhomogeneous, giving rise to anisotropic and non-uniform flow of thermalenergy during conformational fluctuations. To illustrate such features of proteins, we havedeveloped a theoretical framework and computer software (https://curp.jp) for analyzing
local thermal transport properties based on the autocorrelation function formalism. Thismethod has been applied to a wide range of biophysical problems [3–7].
Regarding thermal energy transport mechanism of proteins, there still remains an ongoingdebate as to what determines dominant pathways [8, 9]. Recently, we applied our
computational method to a small protein domain, villin headpiece [9], and evaluatedresidue-wise thermal conductivities [10]. As a result, we observed their distinct residue-type
dependence [11]. Furthermore, we calculated residue-residue thermal conductivities andevaluated the importance of the contact distance, the variance of contact distance, and Hbonding occurrence probability by using the random forest model, a machine learningalgorithm [12].
[1] Eds. DM Leitner, JE Straub, Proteins: Energy, heat, and signal flow, CRC Press (2010).
[2] DM Leitner, T Yamato, Rev. Comput. Chem. 13, 63 (2018).
[3] T Ishikura, T Yamato, Chem. Phys. Lett. 432, 533 (2006)[4] T Ishikura, Y Iwata, T Hatano, T Yamato, J. Comput. Chem. 36, 1709 (2015).
[5] K Ota, T Yamato, J. Phys. Chem. B 123, 768 (2019).
[6] KM Reid, T Yamato, DM Leitner, J. Phys. Chem. B 124, 1148 (2020).
[7] H Poudel, DM Leitner, J. Phys. Chem. B 125, 6522 (2021).
[8] E Deniz, et al. Nat. Commun. 12, 3284 (2021).
[9] S Yamashita, M Mizuno, DP Tran, H Dokainish, A Kitao, Y Mizutani, J.Phys.Chem.B
122, 5877 (2018).
[10] T Yamato, T Wang, W Sugiura, O Laprévote, T Katagiri, J. Phys. Chem. B 126, 3029
(2022).
[11] T. Wang, T. Yamato, W. Sugiura, J. Chem. Phys. 158, 214105 (2023).
[12] T. Wang, T. Yamato, W. Sugiura (to be submitted).