Search

Tounderstand chemical reactivity of molecules in condensed phasein real time, a structural dynamics technique capable of monitoringmolecular conformational motions on their intrinsic time scales, typicallyon femtoseconds to picoseconds, is needed. We have studied a strongphotoacid pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS,pKa* ≈ 0) in pure methanol andobserved that excited-state proton transfer (ESPT) is absent, in sharpcontrast with our previous work on HPTS in aqueous solutions whereinESPT prevails following photoexcitation. Two transient vibrationalmarker bands at ∼1477 (1454) and 1532 (1528) cm–1appear in CH3OH (CD3OD), respectively, risingwithin the instrument response time of ∼140 fs and decayingwith 390–470 (490–1400) fs and ∼200 ps time constantsin CH3OH (CD3OD). We attribute the mode onsetto small-scale coherent proton motion along the pre-existing H-bondingchain between HPTS and methanol, and the two decay stages to the low-frequencyskeletal motion-modulated Franck–Condon relaxation within ∼1ps and subsequent rotational diffusion of H-bonding partners in solutionbefore fluorescence. The early time kinetic isotope effect (KIE) of∼3 upon methanol deuteration argues active proton motions particularlywithin the first few picoseconds when coherent skeletal motions areunderdamped. Pronounced quantum beats are observed for high-frequencymodes consisting of strong phenolic COH rocking (1532 cm–1) or H-out-of-plane wagging motions (952 cm–1)due to anharmonic coupling to coherent low-frequency modes impulsivelyexcited at ca. 96, 120, and 168 cm–1. The vividillustration of atomic motions of HPTS in varying H-bonding geometrywith neighboring methanol molecules unravels the multidimensionalenergy relaxation pathways immediately following photoexcitation,and provides compelling evidence that, in lieu of ESPT, the photoacidityof HPTS promptly activates characteristic low-frequency skeletal motionsto search phase space mainly concerning the phenolic end and to efficientlydissipate vibrational energy via skeletal deformation and proton shuttlingmotions within the intermediate, relatively confined excited-stateHPTS–methanol complex on a solvent-dependent dynamic potentialenergy surface. [ABSTRACT FROM AUTHOR]

Fluorescentproteins (FPs) are luminescent biomolecules that emitcharacteristic hues upon irradiation. A group of calmodulin (CaM)-greenFP (GFP) chimeras have been previously engineered to enable the opticaldetection of calcium ions (Ca2+). We investigate one ofthese genetically encoded Ca2+biosensors for optical imaging(GECOs), GEM-GECO1, which fluoresces green without Ca2+but blue with Ca2+, using femtosecond stimulated Ramanspectroscopy (FSRS). The time-resolved FSRS data (<800 cm–1) reveal that initial structural evolution following 400 nm photoexcitationinvolves small-scale coherent proton motions on both ends of the chromophoretwo-ring system with a <250 fs time constant. Upon Ca2+binding, the chromophore adopts a more twisted conformation in theprotein pocket with increased hydrophobicity, which inhibits excited-stateproton transfer (ESPT) by effectively trapping the protonated chromophorein S1. Both the chromophore photoacidity and local environmentform the ultrafast structural dynamics basis for the dual-emissionproperties of GEM-GECO1. Its photochemical transformations along multidimensionalreaction coordinates are evinced by distinct stages of FSRS spectralevolution, particularly related to the ∼460 and 504 cm–1modes. The direct observation of lower frequencymodes provides crucial information about the nuclear motions precedingESPT, which enriches our understanding of photochemistry and enablesthe rational design of new biosensors. [ABSTRACT FROM AUTHOR]