Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185 .fwdarw. Phe: Formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct

Abstract

Near-infrared FT-Raman spectroscopy can be used to measure the vibrations of the bacteriorhodopsin (bR) chromophore without the disadvantage of conventional visible resonance Raman spectroscopy, where the visible excitation drives the bR photoreactions. We utilized this technique to investigate the light-dark adaptation of bacteriorhodopsin and the mutant Tyr-185-->Phe (Y185F) at room temperature in solution. Compared to wild-type bR, both the FT-Raman and resonance Raman spectra of the light-adapted Y185F displayed new features characteristic of the vibrations of the O intermediate. Light adaptation of Y185F was found to involve a 13-cis, C=N syn-->all-trans isomerization of the retinal chromophore which produces a species similar to bR570 and a second O-like species. Dark adaptation, which was much slower in Y185F compared to wild-type bR, involved a parallel decay of the bR570 and O-like species and resulted in a decreased all-trans:13-cis ratio compared to wild type. Further evidence for the existence of an O-like species in Y185F comes from pump-probe Raman difference spectroscopy, where a red pump beam is found to produce a species very similar to the N intermediate in the photocycle. This species is shown by stroboscopic Raman measurements to exist transiently even at high pH. We postulate that when the Y185F chromophore has an all-trans structure the effective pKa of Asp-85 and Asp-212 is elevated in Y185F due to the disruption of the Asp-212/Tyr-185 hydrogen bond, thereby accounting for the increased protonation of these residues in the O-like species.

Protein Conformational Changes during the Bacteriorhodopsin Photocycle

“…This effect can be explained by the selective photoreaction of the low temperature trapped O-like photoproduct with red light to form an N-like species. A similar effect has been previously observed for the O intermediate formed by the bR mutant Y185F (39). At room temperature, the O photocycle appears to involve a K-like intermediate and a long-lived N intermediate (49,51).…”

“…Resonance Raman Spectroscopy-Resonance Raman spectra were measured using a previously described apparatus (38,39). The sample (250 l, 40 mM Bis-Tris-propane buffer at pH 9.6) was placed in a quartz spinning cell of 19 mm internal diameter and spun with a time period of 22 ms.…”

Characterization of photo-intermediates in the photo-reaction pathways of a bacteriorhodopsin Y185F mutant using in situ photo-irradiation solid-state NMR spectroscopy

“…3A), in which negative peaks indicate reactant species and positive peaks indicate product species. 3B 15 As shown in Fig. 3A indicates that the CS (21.7 ppm) and AT (13.2 ppm) states decreased and the N-(19.2 ppm) and CS*-(18.0 ppm) intermediates increased under irradiation with 520 nm light.…”

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

“…HPLC of the extracted retinal gave 40 and 55% 13-cis content for dark-adapted D85N and D85N͞D96N, respectively, but could give no information on the configuration of the C 15 AN bond (37). Recently FT-Raman (39,40) became the method of choice for determining the configuration of retinal because (i) it does not perturb the protein, (ii) unlike resonance Raman it utilizes 1.064 m excitation that has virtually no effect on the isomeric state, and (iii) it can distinguish between 13-cis,15-anti and 13-cis,15-syn. When the protonation state of the Schiff base is unchanged, the amplitudes of several vibrational modes determine the configuration of the bonds between C 13 and C 15 unambiguously.…”