Effects of detergent environments on the photocycle of purified monomeric bacteriorhodopsin

Abstract

Time-resolved difference spectra have been obtained for the photocycle of delipidated bacteriorhodopsin monomers (d-BR) in six different detergent micelle environments that were prepared by two new detergent-exchange techniques. A global kinetic analysis of the photocycle spectra for d-BR in each detergent environment was performed. Comparison of these results with those obtained for the photocycle of bacteriorhodopsin in purple membrane (PM) shows that there is one fewer kinetically distinguishable process for monomeric BR between the decay of the K intermediate and the rise of the M intermediate. Assuming a sequential pathway occurs in the photocycle, it appears that the equilibrium between the L and M intermediates is reached much more rapidly in the detergent micelles. This is attributed to a more direct interaction between Asp-85 and the proton on the nitrogen of the Schiff base of retinal for BR in the detergents. Equilibrium concentrations of late photocycle intermediates are also altered in detergents. The later steps of the photocycle, including the decay of the M intermediate, are slowed in detergents with rings in their hydrocarbon region. This is attributed to effects on conformational changes occurring during the decay of M and/or other later photocycle intermediates. The lifetime of dark adaptation of light-adapted d-BR in different detergent environments increases in environments where the lifetime of the M intermediate increases. These results suggest that the high percentage of either unsaturated or methyl-branched lipids in PM and the membranes of other retinal proteins may be important for their effective functioning.

Nonionic Homopolymeric Amphipols: Application to Membrane Protein Folding, Cell-Free Synthesis, and Solution Nuclear Magnetic Resonance

“…24 This phenomenon is attributed to a conformational relaxation that would bring the side chain of Asp-85, the residue that accepts the Schiff base's proton, closer to the latter. 36 The rate of buildup of M in NAPols is intermediate between those observed in OTG and A8-35, with a suggestion that it is somewhat less homogeneous, the full transition to M being spread over a slightly larger time scale ( Figure 2). …”

Effects of Asp-96 .fwdarw. Asn, Asp-85 .fwdarw. Asn, and Arg-82 .fwdarw. Gln single-site substitutions on the photocycle of bacteriorhodopsin

“…M decay in d-BR/CHAPSO is drastically slowed down compared to PM and is further slowed down with increasing pH. d-BR/NG, on the other hand, has an M decay rate that is similar to the one in PM, and it is not reduced with increasing pH in the pH range 4-7 (Milder et al, 1991). The effect that replacing Asp-96 with Asn has on the decay of the M intermediate in PM has been attributed primarily to a large change in the entropy of activation for the Schiff base reprotonation in D96N (Miller & Oesterhelt, 1990;Tittor et al, 1989).…”

“…Thus, for each series of time-resolved difference spectra, care was taken to light-adapt all the samples for the same length of time. Dark adaptation rates depend on which detergent is used (Milder et al, 1991). In NG, the dark adaptation rates are relatively fast, and we maintained a low light flux (0.04 W/cm2) on the sample during data collection.…”

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

“…S9). Secondly, the reconstitution of archaeal rhodopsins with detergent or exogenous lipids disturbs their photokinetics in various degrees [90,91], which suggests that a natural lipid environment is important for archaeal rhodopsin to maintain its native structure and function [92][93][94][95]. The lipid environments of the host, strain L33, is thought to facilitate the protein folding of recombinant AR4 since both xz515 and L33 are halophilic Archaea.…”