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 Å 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.
Highlights
Bacteriorhodopsin, a 27-kD membrane protein, functions as a light-driven proton pump in the membranes of the halophilic organism H. salinarum
X-ray analyses from oriented membrane stacks [14,44,45,46], electron paramagnetic resonance (EPR) experiments [47], and several electron microscopic studies [17] have suggested that the movements of helices F and G are involved in such an opening of the cytoplasmic region
The same kind of movement was reported for the N intermediate state after studying an Nintermediate analog, the F219L mutant trapped after illumination, by electron microscopy of tilted 2-dimensional crystals [48]
Summary
Bacteriorhodopsin (bR), a 27-kD membrane protein, functions as a light-driven proton pump in the membranes of the halophilic organism H. salinarum. A single molecule of retinal that is covalently attached to the protein via a protonated Schiff base serves as the chromophore for light absorption. The transmembrane proton gradient generated is available to drive other cellular functions such as transport of other ions or small molecules, synthesis of ATP, or rotation of the flagellar motor for cell motility. Extensive spectroscopic studies have provided a description of the sequence of intermediates generated during the photocycle (Fig. 1) [7,8], while functional analysis of a large number of mutants [9,10,11] had identified key residues that line the path of the proton at different stages of transport
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