Vibrational Spectrum of a Picosecond Intermediate in the Artificial BR5.12 Photoreaction:  Picosecond Time-Resolved CARS of T5.12

Abstract

The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a five-membered carbon ring spanning the C12−C13C14 bonds (BR5.12) is measured by picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). Observed initially by picosecond transient absorption (PTA) measurements, T5.12 is the only intermediate in the BR5.12 photoreaction (i.e., T5.12 decays only to BR5.12). BR5.12 does not have a photocycle analogous to that in native BR, presumably because the five-membered ring blocks the reaction coordinate leading to C13C14 bond isomerization. Since T5.12 may therefore represent the molecular events (reaction coordinates) that precede C13C14 bond isomerization, its vibrational spectrum may aid in elucidating the primary reaction coordinate(s) in the BR photocycle. Although T5.12 is identified via a red-shifted absorption (660 nm maximum, <3 ps formation with 3 ps BR5.12 excitation and decay in 17 ± 1 ps), no spectroscopic data which directly characterize the retinal structure in T5.12, and thereby the role of bonding changes, have been available. The PTR/CARS vibrational data presented here show that T5.12 contains (i) an all-trans retinal configuration, (ii) significant hydrogen out-of-plane motion localized in specific normal modes, (iii) increased π-electron density in the CC stretching modes manifested by frequency increases, (iv) restricted in-plane C−CH3 rocking motion, and (v) a Schiff-base environment similar to that in BR5.12. These PTR/CARS data also confirm that T5.12 decays exclusively to BR5.12. The vibrational spectrum of T5.12 makes it evident that no complete CC isomerization nor C−C rotation in any retinal bond occurs upon excitation of BR5.12. The excellent agreement between the kinetic lifetime of T5.12 (from PTA and PTR/CARS data) and its stimulated emission lifetime suggests that T5.12 may be an excited electronic state. In such a case, the PTR/CARS data presented here are the first to be reported from an excited electronic state of a protein. Regardless of whether T5.12 is an excited or ground electronic state, the vibrational spectra of T5.12 reflect the retinal structure(s) that precedes C13C14 bond isomerization in the BR photocycle. The relevance of T5.12 PTR/CARS data to the native BR photocycle is discussed in terms of the intermediates K-590, J-625, and I-460. Direct analyses of the respective vibrational spectra of T5.12 and K-590 demonstrate that they contain distinctly different retinal structures, but since no vibrational data assignable directly to either I-460 or J-625 have been reported, comparisons of T5.12 with these intermediates are based only on analogy. Comparisons of the vibrational spectra of T5.12 and native BR intermediates independently provide insight into the structural changes in retinal that could occur prior to C13C14 bond isomerization in native BR.

Ultrafast isomerization dynamics of retinal in bacteriorhodopsin as revealed by femtosecond absorption spectroscopy

“…Time-resolved absorption of 13-trans-locked BR has shown similar spectra at 660 nm, just like that of native BR, indicating the all-trans configuration of J-intermediate [17] . Results on the picosecond coherent anti-Stokes Raman Spectroscopy of wild and modified BR illustrated the same case [22,23] . Nevertheless, Herbst and his coworkers [24] have given direct vibrational-spectroscopic evidence for isomerization taking place within 0.5 ps and J-intermediate being 13-cis configuration by studying the vibrational dynamics of the retinal chromophore all-trans-to-13-cis isomerization in BR with mid-infrared absorption spectroscopy at time resolution of 200 fs.…”

Fast spectral coherent anti-Stokes Raman scattering microscopy with high-speed tunable picosecond laser

“…An average laser power >300 mW was high enough for obtaining CARS images with multifocus scanning. 15 Figure 1 shows a schematic diagram of the modified AOTF laser for synchronization with another picosecond mode-locked laser. A prism pair was provided to passively reduce the dispersion of the laser cavity.…”

“…A picosecond mode-locked laser has been used as a narrowband tunable laser source to achieve high spectral resolution. 12,[15][16][17] In biological applications, Raman spectra are usually observed in fingerprint and CH vibrational regions, typically around 1000 and 300 cm −1 , respectively. 10,11,14 Further enhancement can be achieved by extending the observation range to the silent region at 1800 to 2800 cm −1 where some Raman-tag molecules generate signals without any interference from biological specimens.…”

Time-Resolved and Short-Pulse Laser Spectroscopies

“…* Of these techniques, coherent anti-Stokes Ram an spectroscopy has fou nd the most general use. [23][24][25] Because of the high peak power requirements of such measurem ents, picosecond lasers have been used widely, and picosecond lasers hold an advantage in terms of linewidth matching and heating. Recent measurements with the use of picosecond CARS spectroscopy have focused on gaining a m echanistic und ers tand ing of the photoinduced dynamics of bacteriorhodopsin, the compound central to vision.…”

“…The initial step in the dynamical response of this molecule is thought to be isomerization about the C 13 5C 14 double bond, and CARS is ideally suited to m easure the transient vibrational response of this system. 23 In addition to studies on rhodopsin, the isom erization of other molecules is amenable to examination by CARS, even for scattering from excited electronic states. 25 En- …”