Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses

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

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release