Probing the early stages of photoreception in photoactive yellow protein with ultrafast time-domain Raman spectroscopy

Abstract

Unveiling the nuclear motions of photoreceptor proteins in action is a crucial goal in protein science in order to understand their elaborate mechanisms and how they achieve optimal selectivity and efficiency. Previous studies have provided detailed information on the structures of intermediates that appear during the later stages (>ns) of such photoreception cycles, yet the initial events immediately after photoabsorption remain unclear because of experimental challenges in monitoring nuclear rearrangements on ultrafast timescales, including protein-specific low-frequency motions. Using time-domain Raman probing with sub-7-fs pulses, we obtain snapshot vibrational spectra of photoactive yellow protein and a mutant with high sensitivity, providing insights into the key responses that drive photoreception. Our data show a drastic intensity drop of the excited-state marker band at 135 cm within a few hundred femtoseconds, suggesting a rapid weakening of the hydrogen bond that anchors the chromophore. We also track formation of the first ground-state intermediate over the first few picoseconds and fully characterize its vibrational structure, revealing a substantially-twisted cis conformation.

Two-Dimensional Impulsively Stimulated Resonant Raman Spectroscopy of Molecular Excited States

“…Furthermore, Raman spectroscopy benefits from resonance enhancements of specific chromophore signatures and thereby provides a route to comprehensively investigate vibrational energy flow during reactive transformations by selectively probing specific environments [25]. In particular, time-domain impulsive stimulated Raman scattering (ISRS) [26][27][28][29][30][31][32][33][34][35][36] offers several advantages over its frequency-domain analogues for the detection of vibronic features, especially for low-frequency modes, by efficiently removing elastic scattering contributions and background noise [37][38][39][40]. Its multidimensional extension, 2D-ISRS, has been theoretically proposed initially [41] and realized in both nonresonant [42,43] and resonant [44,45] implementations to study ground-state intramolecular vibrational anharmonicities, nonlinear corrections to the molecular polarizability, product-reactant correlations, and solvation dynamics, up to the recent realization of single-pulse 2D spectroscopy by means of appropriately shaped light pulses [46], with possible applications theoretically suggested for the x-ray domain [47].…”

“…Here, we address these challenges by presenting resonant excited-state 2D-ISRS. In striking contrast with the time-resolved ISRS approach-which employs a photopump to create an electronically excited-state population and monitors the temporal evolution of vibrational frequencies during a photoreaction [30,32,49]-by exploiting three femtosecond pulses for stimulating Raman coherences, we induce and probe vibronic correlations on the electronically excited manifold. Building on the resonance Raman enhancement, in our realization we tune the optical wavelengths of the pulses used in 2D-ISRS in resonance with the static and transient electronic absorption transitions to isolate contributions pertaining to a targeted electronic state.…”

Photoinduced Structural Dynamics of Molecular Systems Mapped by Time-Resolved X-ray Methods

“…The structural changes of the p-coumaric acid chromophore and the protein were recorded down to 100 fs. 348 These measurements probed the motions of the early Franck-Condon (FC) excited state that has recently been also studied by time-resolved Raman spectroscopy at high temporal resolution (<7 fs) 349 showing that a rapid weakening of the hydrogen bond that anchors the chromophore is the primary event out of the FC region.…”

Spectroscopic ruler for measuring active-site distortions based on Raman optical activity of a hydrogen out-of-plane vibration

“…Proposed functional roles for cofactor distortions include the out-of-plane distortion of chromophores as a key factor in controlling their absorption spectra (3,4). Furthermore, photoexcitation of these proteins produces primary high-energy intermediates with structurally perturbed chromophores (5)(6)(7)(8), which drive subsequent protein conformational changes (9,10). Such structural distortions have proven difficult to measure experimentally.…”