1C17
Unraveling the excited-state structural dynamics of LSSmOrange protein
through time-resolved impulsive stimulated Raman spectroscopy○ Pardeep Kumar1,2, David Manuel Rantasa3, Eduard Fron4, Haruko Hosoi5,Hikaru Kuramochi1,2,6, Satoshi Takeuchi1,2, Hideaki Mizuno3, Tahei Tahara1,2
1
Molecular Spectroscopy Laboratory, RIKEN, Japan
2
RIKEN Center for Advanced Photonics (RAP), RIKEN, Japan
3
Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and StructuralBiology Section, Department of Chemistry, KU Leuven, Belgium
4
Laboratory of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven,Belgium
5Department of Biomolecular Science, Faculty of Science, Toho University, Japan
6JST PRESTO, Japan[Abstract] LSSmOrange is a mutant of a coral fluorescent protein, DsRed. The absorption
and emission maxima of LSSmOrange are separated by 5453 cm-1, and this large energy
difference has provided the capability of multicolor imaging with a single excitation laser line
in living cells. The chromophore of LSSmOrange has a neutral form in the ground state. Upon
photoexcitation, it undergoes ultrafast excited-state proton transfer (ESPT), resulting in theformation of the bright anionic state. This ESPT dynamics of the LSSmOrange is represented
by the multi-exponential kinetics, however, its mechanism has not been fully understood yet.
To clarify the ESPT mechanism from a structural viewpoint, we employed time-resolved
impulsive stimulated Raman spectroscopy (TR-ISRS) using sub-10 fs pulses, and recorded
the Raman spectra of the chromophore with femtosecond time resolution. The obtained
time-resolved Raman data indicate that the chromophore in LSSmOrange is present in two
different structural forms and thus explain the observed multi-exponential kinetics of theESPT.
[Introduction] Studying biological processes in the living cells is a challenging task, however,Fig. 1. (A) Chromophore structure with interactions with the nearest amino acids in LSSmOrange based on
the crystallographic structure (PDB ID: 4Q7R). Blue and orange traces are the steady state absorption andfluorescence spectra of LSSmOrange (pH 7.4, 20 mM Tris-HCl buffer + 150 mM NaCl). (B) Measurement
scheme and pulse sequence of TR-ISRS. Blue and orange traces denote a pump-probe signal measured withP1 and P3 pulses, and a TR-ISRS signal measured with P1, P2 and P3 pulses, respectively. Green trace isthe time-domain Raman signal, which is extracted from the TR-ISRS signal by subtracting theslowly-varying population component.

fluorescent proteins have provided the capability of observing these biological processes
under the physiological conditions. LSSmOrange is a fluorescent protein developed to fill thespectral gap between green-yellow and red fluorescent proteins. The absorption and emission
maxima of LSSmOrange is separated with very large energy (Figure 1A). This large energy
separation is rationalized with the mechanism of ultrafast excited-state proton transfer(ESPT)1. The initial photo-excited chromophore has a neutral form (Figure 1A), and it
undergoes the ultrafast ESPT, resulting in the formation of the bright anionic state. However,
the excited-state dynamics of LSSmOrange have not been clearly understood yet. To clarify
the excited-state dynamics from a structural viewpoint, we employed time-resolved impulsive
stimulated Raman spectroscopy (TR-ISRS)2 using sub-10 fs pulses, and recorded the Raman
spectra of the chromophore with femtosecond time resolution.
[Methods] Measurement scheme and pulse sequence of TR-ISRS are shown in Figure 1B.
The actinic pump pulse (P1, 415 nm, 90 fs)
was generated by frequency-doubling theoutput of the Ti:Sapphire amplifier. TheRaman pump and probe pulses (P2 and P3,600–800 nm) were generated using a
non-collinear optical parametric amplifier(NOPA). The output of the NOPA wascompressed to <8 fs using a folded prism
pair and micro-machined membrane
deformable mirror, and the obtained pulse
was characterized by self-diffraction
frequency-resolved
opticalgating(SD-FROG) at the sample position. These
three pulses (P1, P2, and P3) were focused
together onto a 0.3-mm thick flow cell.
[Results and Discussion] Time-resolved
Raman spectra of LSSmOrange, obtainedby TR-ISRS, are shown in Figure 2.
Immediately after photoexcitation (forexample, ΔT = 0), weak Raman bands of Fig. 2. Time-resolved Raman spectra of LSSmOrange
the neutral form before the ESPT were (pH 7.4, 20 mM Tris-HCl buffer + 150 mM NaCl) atobserved.
Subsequently,
onthe various P1-P2 delays (ΔT). Raman spectrum at 0 ps is 10
times magnified (dotted curve).
femto-to-picosecond time scale, weobserved the growth of numbers of Raman bands, which is attributable to the formation of theanionic form chromophore through the ESPT. The kinetic analysis of these anionic form
Raman bands reveals bi-exponential growth of its population. The analysis also indicates that
this bi-exponential feature is associated with two parallel ESPT pathways producing two
different anionic form species with different Raman spectra, suggesting the heterogeneity of
the chromophore structure of LSSmOrange. On the basis of the previous X-ray
crystallographic study of LSSmOrange3, we attribute the origin of this heterogeneity to thedifferent hydrogen bond environments around the chromophore.
[References][1] E. Fron et al. J. Phys. Chem. B, 119, 14880 (2015).
[2] H. Kuramochi et al. Rev. Sci. Instrum., 87, 043107 (2016).
[3] S. Pletnev et al. PLOS ONE, 9, e99136 (2014).