Two-Photon Absorption of Bacteriorhodopsin: Formation of a Red-Shifted Thermally Stable Photoproduct F620

Abstract

By means of high-intensity 532 nm laser pulses, a photochemical conversion of the initial B(570) state of bacteriorhodopsin (BR) to a stable photoproduct absorbing maximally at approximately 620 nm in BR suspensions and at approximately 610 nm in BR films is induced. This state, which we named F(620), is photochemically further converted to a group of three products with maximal absorptions in the wavelength range from 340 nm to 380 nm, which show identical spectral properties to the so-called P(360) state reported in the literature. The photoconversion from B(570) to F(620) is most likely a resonant two-photon absorption induced step. The formation of F(620) and P(360) leads to a distinguished photo-induced permanent optical anisotropy in BR films. The spectral dependence of the photo-induced anisotropy and the anisotropy orientations at the educt (B(570)) and product (F(620)) wavelengths are strong indicators that F(620) is formed in a direct photochemical step from B(570). The chemical nature of the P(360) products probably is that of a retro-retinal containing BR, but the structural characteristics of the F(620) state are still unclear. The photo-induced permanent anisotropy induced by short laser pulses in BR films helps to better understand the photochemical pathways related to this transition, and it is interesting in view of potential applications as this feature is the molecular basis for permanent optical data storage using BR films.

Two Bathointermediates of the Bacteriorhodopsin Photocycle, from Time-Resolved Nanosecond Spectra in the Visible

“…At such levels of energy density any possible photochemistry will be overshadowed by trivial thermal damage, and the sample would be burned. These considerations results in a well-known rule of thumb: it is next to impossible to saturate a transition that occurs on a time-scale much shorter than the pulse-length used. Since under nanosecond excitation photodamage becomes pronounced only when excitation energy density approaches or exceeds saturation intensity (see the results in 86 , which are confirmed by our own observations), the responsible state should decay on a nanosecond but not picosecond or faster time-scale. Therefore, in the view of the kinetics discussed above, the K E state seems to be the only candidate.…”

“…Since under nanosecond excitation photodamage becomes pronounced only when excitation energy density approaches or exceeds saturation intensity (see the results in 86 , which are confirmed by our own observations), the responsible state should decay on a nanosecond but not picosecond or faster time-scale. Therefore, in the view of the kinetics discussed above, the K E state seems to be the only candidate.…”

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin in the multiphoton regime and biological relevance

“…However, we estimate that the fully resonant 2-photon absorption of retinal at 535 nm is at least 2 orders of magnitude larger than the nonresonant process for Trp 10,11 . This estimate is further reinforced by the observed saturation of the nonresonant 2-photon absorption (800 nm) of retinal (bR) at 700 GW/cm 2 -to give a completely different photoproductthat is specifically exploited for optical memory 12,13 . The fully resonant 2-photon absorption of retinal (at 535 nm) would saturate at more than an order of magnitude lower peak power than observed at 800 nm, approximately where the authors see the onset of nonlinear behavior (>30 GW/cm 2 , Fig.…”

Resonant Two-Photon Excitation Pathways During Retinal-Isomerization in Bacteriorhodopsin

“…This suggests that the red-absorbing intermediate found on a pico-to nanosecond timescale is not a stationary product and that additional evolution occurs on longer timescales (> 100 ns). Hence, the red-absorbing photoproduct can not be the same as two-photon induced photoproducts such as the Laser-induced blue-membrane-(LIBM) [3] or the F 620 -state [4], which are observed for high-energy nanosecond excitation. Our results thus suggest that additional two-photon induced photoproducts can be generated by sub-30 fs excitation directly from the Franck-Condon point and that the LIBM and F 620 photoproducts [3][4] are generated from later species in the photocycle.…”