Restriction of motion of protein side chains during the photocycle of bacteriorhodopsin.

Abstract

Linear dichroism was measured during the photocycle of bacteriorhodopsin. The anisotropy of the sample was produced by the photoselection method. The measurements on purple membrane fragments embedded in agar gel were performed at room temperature with 200-psec time resolution at several wavelengths in the 240-to 550-nm spectral region. The induced anisotropy of the retinal chromophore remained constant after the formation of the photocycle intermediate M. The anisotropy was also time independent at the characteristic peaks ofthe UV absorption change. These-experimental data suggest that the direction of the retinal transition dipole moment remains unchanged. Moreover, the affected aromatic protein side chains also do not show any rotational motion when they are in the perturbed or ground states during the photocycle. Our data render it possible to calculate the restricted range of sudden chromophore rotations that might be coupled to the appearance and decay of the M intermediate.A study of chromophore orientation in bacteriorhodopsin (bR) during its photocycle can provide valuable information about the proton-pumping mechanism of this molecule. Photoselection is a convenient method for this purpose. Photoselection measurements on bR have been reported only in the visible range, however (1-9).Slow decay of the induced dichroism of the retinal chromophore at different wavelengths caused by the rotational diffusion ofpurple membranes (PMs) has been observed in aqueous solution (4,5,8). Small, fast changes in the polarization anisotropy also have been reported and attributed to some conformational changes ofthe protein during the photocycle (4, 5). These transients disappeared, however, when PMs were immobilized in polyacrylamide gel, indicating that the fast decay was probably due to rotational diffusion of small membrane fragments (10). Transient dichroism data obtained on bR incorporated into phosphatidylcholine vesicles (9) have shown that the bRs are immobilized below the lipid phase transition temperature and rotate above it. The slow transition of light-adapted bR to the dark-adapted form has been measured by the photoselection method at 530 nm on-thin layers containing PM (11). This experiment indicated that both the bR molecules in the PM and the visible retinal chromophore in the ground state are immobilized. Recently, however, changes -of the photodichroism in the visible range during the photocycle have been measured and interpreted as a result ofrotational displacement ofthe retinal chromophore (7).The spectrum ofthe UV absorption change indicates that the aromatic amino acid residues of the protein participate in the proton-pumping mechanism (12-14). These transients in films containing PM have been found to be dichroic, suggesting that only few residues are involved (14). The formation and decay times have been correlated with the steps ofthe photocycle (15, 16).Here we report photoselection measurement on PM performed in a wide wavelength range (240-550 nm). PMs were immobilized in ==1% ag...

Light-induced reorientation in the purple membrane

“…The observation of reorientational motion triggered by light absorption and consequently correlated with the BR photocycle suggests that these reorientational motions might play a functional role in the proton-pumping mechanism of the purple membrane. These motions, however, are not essential to the proton-pumping mechanism, since monomeric BR and BR immobilized in gels are still capable of pumping protons (21,22). If the reversible rotational model with reorientation of spectator BR monomers is correct, the role of these reorientations may well be related to the puzzling question:…”

“…Orientational motion in the PM has also been investigated, principally by linear dichroism spectroscopy, which detects reorientation of the retinal chromophore following polarization-selective excitation of BR (18)(19)(20)(21)(22)(23). Some of these studies have detected reorientation in BR during the M--N-*O->BR transition (18,19,22,23).…”

“…Ahl and Cone (22) attributed orientational motion observed with -1-ms resolution in PM suspensions to the rotation of monomeric proteins within the trimer in the BR membrane. Studies of BR in gels (agar or polyacrylamide), on the other hand, have not detected orientational motion (21,24,25).…”

Direct Measurement of the Photoelectric Response Time of Bacteriorhodopsin via Electro-Optic Sampling

“…But none of the devices studied to date tests the ultimate capability of the protein to provide an ultrafast response. To establish the full potential of such integrated devices and circuits for high speed applications, it is necessary to characterize the dynamics of the initial (B1) photovoltage transient (Blaurock and Stoeckenius, 1971;Khorana et al, 1979;Czege et al, 1982;Kononenko et al, 1987;Henderson et al, 1990). This study determines the initial (fast) photovoltaic response of the protein by using a technique with an effective 3-dBbandwidth of 350 GHz, a temporal resolution roughly six times greater than previous studies.…”

Reply to “Comment on ‘An Apparent General Solution for the Kinetic Models of the Bacteriorhodopsin Photocycle' ”

“…If repeated determinations find that there is a small but significant increase in the slowest time constant for membranes encased in gel, a likely explanation is that the recovery of BR from M entails the greatest conformational change of the bR or membrane, as indicated by the anisotropy values of Song et al 6 and the high viscosity of the gel impedes this transition. The idea that there is significant rotational diffusion of PM in suspensions that can lead to distortions of kinetic data emanating from reorientations of the retinal chromophore in photoselected populations seemed to be supported by Czege et al 8 who reported that embedding PM in agar gels removed the changes in anisotropy observed in suspensions. They concluded "the direction of the retinal chromophore (and consequently of the retinal itself) does not change even during the transitions of the photocycle."…”