Dataset Information

Protein conformational changes in the bacteriorhodopsin photocycle: comparison of findings from electron and X-ray crystallographic analyses.

ABSTRACT: Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 A resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes.

SUBMITTER: Hirai T

PROVIDER: S-EPMC2685002 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Publications

Protein conformational changes in the bacteriorhodopsin photocycle: comparison of findings from electron and X-ray crystallographic analyses.

Hirai Teruhisa T Subramaniam Sriram S

PloS one 20090602 6

Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structur ...[more]

PMID: 19488399

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Project description:It is becoming increasingly clear that the transfer of an electron across a protein-protein interface is coupled to the dynamics of conformational conversion between and within ensembles of interface conformations. Electron transfer (ET) reactions in conformationally mobile systems provide a "clock" against which the rapidity of a dynamic process may be measured, and we here report a simple kinetic (master equation) model that self-consistently incorporates conformational dynamics into an ET photocycle comprised of a photoinitiated "forward" step and thermal return to ground. This kinetic/dynamic (KD) model assumes an ET complex exists as multiple interconverting conformations which partition into an ET-optimized (reactive; R) population and a less-reactive population ( S). We take the members of each population to be equivalent by constraining them to have the same conformational energy, the same average rate constant for conversion to members of the other population, and the same rate constants for forward and back ET. The result is a mapping of a complicated energy surface onto the simple "gating", two-well surface, but with rate constants that are defined microscopically. This model successfully describes the changes in the ET photocycle within the "predocked" mixed-metal hemoglobin (Hb) hybrid, [alpha(Zn), beta(Fe3+N 3 (-))], as conformational kinetics are modulated by variations in viscosity (eta = 1-15 cP; 20 degrees C). The description reveals how the conformational "routes" by which a hybrid progresses through a photocycle differ in different dynamic regimes. Even at eta = 1 cP, the populations are not in fast exchange, and ET involves a complex interplay between conformational and ET processes; at intermediate viscosities the hybrid exhibits "differential dynamics" in which the forward and back ET processes involve different initial ensembles of configurational substates; by eta = 15 cP, the slow-exchange limit is approached. Even at low viscosity, the ET-coupled motions are fairly slow, with rate constants of <10 (3) s (-1). Current ideas about Hb function lead to the testable hypothesis that ET in the hybrid may be coupled to allosteric fluctuations of the two [alpha 1, beta 2] dimers of Hb.

Dataset Information

Protein conformational changes in the bacteriorhodopsin photocycle: comparison of findings from electron and X-ray crystallographic analyses.

Publications

Protein conformational changes in the bacteriorhodopsin photocycle: comparison of findings from electron and X-ray crystallographic analyses.

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