Nanosecond retinal structure changes in K-590 during the room-temperature bacteriorhodopsin photocycle: picosecond time-resolved coherent anti-stokes Raman spectroscopy

Abstract

Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.

Low-Temperature FTIR Study of Multiple K Intermediates in the Photocycles of Bacteriorhodopsin and Xanthorhodopsin

“…The fourth band at ~985 cm −1 , which we assign exclusively to K E , is not expected to be strong and is not seen at 77K in Raman 36,94 but was observed, in accord with our expectations, at room temperature in nanosecond time-resolved CARS 32 and Raman 22 at 984 cm −1 and at 987 cm −1 , respectively. The last two bands at 963 and 997 cm −1 in bacteriorhodopsin and at 954 and 992 cm −1 in xanthorhodopsin are present in both K 0 and K E states and exhibit no temperature dependence (Figure 4A).…”

“…The last of the K states, K L , decays into L with a time constant of ~ 1.2 μs 83 . Two additional transitions were reported within the life-time of the K-like states with the time constants of (i) 30-70 ps 23,31,38 , and (ii) 50-250 ns 16,28,32 , respectively. We assign the former, with the τ 1 =50±20 ps time constant, to the K 0 -to-K E transition; the latter, with the τ 2 =150±100 ns time constant, had been already assigned to K E -to-K L transition 16,28 .…”

“…The existence of more than one K-like intermediate is supported by evidence from the variety of experimental methods: in visible 14,16-20 , UV 21 , and FTIR spectroscopy 15,22-28 , resonance Raman 29-31 , CARS 32 , and photoelectric measurements 33 . The necessity for more than one K-like intermediates has been evident since early eighties 14,15 but the question on their number has not been settled, and most authors restrict their discussions to the possibility of two different K-like states 14-18,21-31 .…”

“…The vibrational spectra of the K-like state(s) have been studied in detail, by resonance Raman 29,30,35-39 , CARS 32,40 , and IR methods 15,23,41-45 , using static (i.e. trapped at cryogenic temperature) 15,35,41-44,46-48 and time-resolved 23-30,36-40,45 techniques.…”

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

“…The calculated IR spectra 29 indicated that the absorption maximum of the K L state is not as strongly bathochromically shifted, as the maximum of K E . Existence of K E and K L as separate intermediates was further supported by time-resolved CARS 33 , in which both the rate constant for the K E -to-K L transition and the main vibrational features of the K E and the K L were shown to be consistent with those in 29 .…”