Picosecond time-resolved ultraviolet resonance Raman spectroscopy of bacteriorhodopsin: primary protein response to the photoisomerization of retinal.

Protein dynamics in the primary processes during the bacteriorhodopsin (BR) photocycle under physiological conditions were investigated by measuring picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of the BR suspended solution at ambient temperature. We used a 565 nm pump pulse to initiate the BR photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, that is, Trp86, Trp182, Tyr57, and Tyr185. The 225 nm UVRR spectra exhibited bleaching of intensity for all the tryptophan bands within the instrumental response, followed by recovery with a time constant of 30 ps and no further changes up to 1 ns. This suggests that the stepwise structural changes in the tryptophan residues proceed in response to the retinal photoreaction. It is concluded that the initial intensity bleach arises from the J-intermediate formation and the 30 ps recovery is associated with the K-KL transition. The 30 ps process in the BR photocycle has been detected for the first time. In the 238 nm UVRR spectra, spectral features attributable to the K and KL intermediates were observed. The observed spectral changes showed that the temporal behaviors of the observed spectral changes in each Raman band of both tryptophan and tyrosine were different. This indicates that the spectral changes originated from structural changes of at least two tryptophan and two tyrosine residues.