Time‐resolved resonance Raman studies of bacteriorhodopsin and its intermediates K₅₉₀ and L₅₅₀: Biological implications

Abstract

Experiments with a spinning cell and a single continuous-wave laser beam (514 nm) in reflection geometry were performed to obtain the resonance Raman (RR) spectra of the intermediate K,,, of the retinal chromophore of bacteriorhodopsin (bR). Samples of diluted aqueous suspensions of 'purple membranes' from Halobacterium halobium were used, which entail bR as an integral protein. K,,, is formed photochemically in CQ. 5 ps from the parent chromophore BR,,, and in H,O decays into the product L , , , with a time constant of ca. 1.2 ps (21 "C). Using a flow velocity of ca. 20 m s-' and a beam waist of 40 pm, the integrated relative concentrations of BR,,, , K , , , and L,,, in the beam were 58: 33:10, respectively. By this method the 'late' K intermediate which is accumulated on a time scale of ea. 1 ps was probed. The spectra of the mixture were recorded with a spectral step width of 0.2 cm-' and a band width of 4 cm-'. Up to 100 scans were accumulated to obtain the high quality which is required for subtraction and band-fitting procedures. The contributions of BR,,, and L,,, , whose spectra were obtained in separate experiments, were subtracted leaving the pure spectra of K,,, . Experiments were performed in H,O and D,O suspensions. Structural changes of the chromophore during the transition from K,,, to L,,, were inferred from the RR spectra in the hydrogen out-of-plane, the fingerprint and the C=C/C=N stretching regions. It was concluded that in this transition the chromophore relaxes from a distorted to a planar conformation and at the same time a positive charge of the protein envelope approaches the fi-ionone ring of retinal. It is proposed that this electrostatic interaction is important for the biological function of bR.

Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: A theoretical analysis of structural elements

“…The present gas-phase calculations indicate that moderate bond twisting (10°-30°) and combined single-and double-bond twisting, as usually observed in the protein environment are unlikely to be the major determinant of the intrinsic affinity for protons of the retinal Schiff base. This finding is consistent with the observations from spectroscopy that the retinal conformation in L, where the Schiff base is ready to deprotonate, is similar to the N-state twist, where the Schiff base becomes again protonated (Lohrmann and Stockburger, 1992), and, moreover, the strength of the hydrogen-bonding interactions between the retinal Schiff base and the protein is similar in L and N (Pfefferlé et al, 1991). A more pronounced twist around the C 14 AC 15 single-bond in L as compared to N (Rö dig et al, 1999) could fine-tune the Schiff base proton affinity.…”

Time‐resolved resonance Raman spectroscopy of sensory rhodopsin II in the micro‐ and millisecond time range using gated cw excitation

“…The averaged photochemical rate constant l 0,P is calculated by integrating the expression of l t for a Gaussian beam shape, 12,13 …”

Vibrational Spectrum of K-590 Containing13C14,15Retinal: Picosecond Time-Resolved Coherent Anti-Stokes Raman Spectroscopy of the Room Temperature Bacteriorhodopsin Photocycle

“…22 Without independently understanding the influences of the protein on the retinal, it is difficult to uniquely assign (interpret) shifts in the CdC and/or C-C stretching frequencies in terms of structural changes in the retinal and/or the protein alone. These data and calculations, therefore, cannot accurately describe the interactions between the retinal and specific opsin sites that are likely to influence the retinal structure.…”