Unraveling Ultrafast Photoinduced Proton Transfer Dynamics in a Fluorescent Protein Biosensor for Ca²⁺ Imaging

Abstract

Imaging Ca(2+) dynamics in living systems holds great potential to advance neuroscience and cellular biology. G-GECO1.1 is an intensiometric fluorescent protein Ca(2+) biosensor with a Thr-Tyr-Gly chromophore. The protonated chromophore emits green upon photoexcitation via excited-state proton transfer (ESPT). Upon Ca(2+) binding, a significant population of the chromophores becomes deprotonated. It remains elusive how the chromophore structurally evolves prior to and during ESPT, and how it is affected by Ca(2+) . We use femtosecond stimulated Raman spectroscopy to dissect ESPT in both the Ca(2+) -free and bound states. The protein chromophores exhibit a sub-200 fs vibrational frequency shift due to coherent small-scale proton motions. After wavepackets move out of the Franck-Condon region, ESPT gets faster in the Ca(2+) -bound protein, indicative of the formation of a more hydrophilic environment. These results reveal the governing structure-function relationship of Ca(2+) -sensing protein biosensors.

Femtosecond Stimulated Raman Spectroscopy

“…Pixel resolution is not a limitation for CCD sensors, which, in addition, offer low noise levels. This makes them the most common detector type used for FSRS experiments . A comparison of different detectors including a discussion of the relevant parameters for FSRS, like dynamic range and readout rate, has been given by Marx et al …”

Femtosecond stimulated Raman line shapes: Dependence on resonance conditions of pump and probe pulses

“…The GS Raman measurements were performed using our home-built table-top FSRS setup with broad wavelength tunability and the details can be found elsewhere [5,12,13,27]. In brief, the Raman pump and probe pulses are generated from the ∼2 W (half of the 4 W output), 800 nm fundamental pulse with 35 femtosecond (fs) duration of a mode-locked Ti:sapphire oscillator and regenerative laser amplifier (Legend Elite-USP-1K-HE, Coherent, Inc.) at 1 kHz repetition rate.…”

“…In the past decade, femtosecond stimulated Raman spectroscopy (FSRS) has become a powerful spectroscopic methodology that can provide the equilibrium and non-equilibrium vibrational signatures and track excited-state molecular dynamics with simultaneously high spectral and temporal resolutions [1][2][3][4][5][6][7]. Over recent years, a great variety of chemically and biologically relevant systems have been studied by FSRS spanning from organic photoacids and chromophores [8][9][10][11][12][13][14][15], molecular rotors [16], fluorescent proteins [3,17], photoreceptor proteins [18][19][20][21][22][23][24][25][26], calcium biosensors [4,[27][28][29], metal complexes [30,31], materials [32][33][34], and engineered molecular systems [35,36]. The underlying photophysical and photochemical processes including excited-state proton transfer, charge transfer, vibrational cooling, internal conversion, isomerization, and bond dissociation have been successfully revealed and discussed in the larger context of effectively delineating the structure-energy-function relationships [6,7].…”

Sum-Frequency-Generation-Based Laser Sidebands for Tunable Femtosecond Raman Spectroscopy in the Ultraviolet

“…The FSRS technology has been successfully applied to a number of important photosensitive molecular systems including rhodopsin [27], bacteriorhodopsin [28], phytochrome [29], organic dyes in solar cells [30], Fe(II) spin crossover in solution [31], fluorescent proteins [7,32,33], and calcium-ion-sensing protein biosensors [34][35][36]. The main goal of this work is to construct a versatile, tunable FSRS setup that extends the wavelength detection window to the UV regime with desired resonance Raman enhancement.…”