Abstract
X-ray and electron diffraction studies of specific reaction intermediates, or reaction intermediate analogues, have produced a consistent picture of the structural mechanism of light-driven proton pumping by bacteriorhodopsin. Of central importance within this picture is the structure of the L-intermediate, which follows the retinal all-trans to 13-cis photoisomerization step of the K-intermediate and sets the stage for the primary proton transfer event from the positively charged Schiff base to the negatively charged Asp-85. Here we report the structural changes in bacteriorhodopsin following red light illumination at 150 K. Single crystal microspectrophotometry showed that only the L-intermediate is populated in three-dimensional crystals under these conditions. The experimental difference Fourier electron density map and refined crystallographic structure were consistent with those previously presented (Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2000) Nature 406, 645-648; Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2001) Photochem. Photobiol. 74, 794-804). Based on the refined crystallographic structures, molecular dynamic simulations were used to examine the influence of the conformational change of the protein that is associated with the K-to-L transition on retinal dynamics. Implications regarding the structural mechanism for proton pumping by bacteriorhodopsin are discussed.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The spectral analysis, difference Fourier map, and refined crystallographic structure demonstrate that the light-induced structural changes previously reported [1] were correctly interpreted as characterizing the build up of low temperature Lintermediate (LLT). It follows that the mechanistic model of vectorial proton transport by bR should incorporate the fact that significant rearrangements of water molecules on the EC half of the protein, a reorientation of the guanidinium group of Arg-82 toward the EC medium, and a local flex of helix C toward the proton translocation channel centered near Asp-85 all occur after retinal photoisomerization yet prior to the primary proton transfer event from the Schiff base to Asp-85
In contrast to the spectral results from three-dimensional crystals at 170 K [1, 2, 15, 16], there is no positive feature at 410 nm in either difference spectrum, which would indicate a contribution from the low temperature M-intermediate (MLT)
Summary
The atomic coordinates and structure factors (code 1R3P) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). The spectral analysis, difference Fourier map, and refined crystallographic structure demonstrate that the light-induced structural changes previously reported [1] were correctly interpreted as characterizing the build up of LLT It follows that the mechanistic model of vectorial proton transport by bR should incorporate the fact that significant rearrangements of water molecules on the EC half of the protein, a reorientation of the guanidinium group of Arg-82 toward the EC medium, and a local flex of helix C toward the proton translocation channel centered near Asp-85 all occur after retinal photoisomerization yet prior to the primary proton transfer event from the Schiff base to Asp-85. Building upon the refined structural models, molecular dynamic simulations are presented that illustrate how the geometry of the retinal is perturbed by the conformational change in the protein associated with the K-to-L transition, providing structural insight into how the L-intermediate sets the stage for the primary proton transfer from the Schiff base to Asp-85 in the L-to-M transition
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