Mechanism of Proton Transport in Bacteriorhodopsin from Crystallographic Structures of the K, L, M1, M2, and M2′ Intermediates of the Photocycle

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Abstract

We produced the L intermediate of the photocycle in a bacteriorhodopsin crystal in photo-stationary state at 170 K with red laser illumination at 60% occupancy, and determined its structure to 1.62 Å resolution. With this model, high-resolution structural information is available for the initial bacteriorhodopsin, as well as the first five states in the transport cycle. These states involve photo-isomerization of the retinal and its initial configurational changes, deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. The six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base.

Introduction

The light-driven proton pump, bacteriorhodopsin, is currently the best understood transmembrane ion transport system.1., 2. The transport cycle (“photocycle”) of this small retinal-protein is the linear reaction sequence of the K, L, M, N and O intermediates and their sub-states.3 The events after absorption of a photon can be divided into two phases: (i) the K–L–M1–M2–M2′ sequence in which the Schiff base of the photoisomerized 13-cis retinal donates its proton, in sequentially shifting equilibria toward complete transfer, to its counter-ion Asp85 and a proton is released to the extracellular surface; and (ii) the N–N′–O–BR sequence after Asp96 reprotonates the Schiff base, in which proton uptake from the cytoplasmic surface reprotonates Asp96 to produce N, the retinal re-isomerizes to all-trans, and a proton is transferred from Asp85 to the proton release site.

The intermediates and the reactions of the photocycle have been described by static and time-resolved visible,3 Raman,4 FTIR5., 6., 7. and NMR8 spectroscopy utilizing a large number of site-specific mutations, and recently also by electron and X-ray crystallography. X-ray diffraction of 3D bacteriorhodopsin crystals, grown with the cubic phase method,9 has yielded a structural description of the non-illuminated protein to 1.90 Å,10 1.55 Å11 and 1.47 Å12 resolution. Illuminating crystals of the wild-type protein and mutants at temperatures between 100 K and 295 K produced photo-stationary states, from which structural changes in the K, L, and the various M states have been reported.12., 13., 14., 15., 16., 17., 18., 19. Cryo-electron microscopy of illuminated 2D crystals, at lower resolution, described an outward tilt of the cytoplasmic end of helix F in the N intermediate.20 Structures for a late M and the O intermediates,21., 22. also with large-scale conformational changes of helices, were proposed from crystallographic descriptions of two mutants in their non-illuminated states.

The structural transformations of the retinal, together with the protein and water in its immediate vicinity, obviously play the key role in the way the steps leading toward thermal re-isomerization of the retinal drive the transport. These changes are subtle, and in several cases need to be extracted from diffraction data after only partial conversion to the intermediate of interest. For these reasons, high crystallographic resolutions (1.43 Å) have been helpful in solving the structures of the K and the M1 states at occupancies of 40% and 60%, respectively,12., 19. We now report on the structure of the L state, at 1.62 Å resolution and 60% occupancy. The structural models for the M2 and M2′ states15., 16. were determined earlier from diffraction data to 1.8 Å and 2.0 Å, respectively, but after full conversion to these intermediates. Together with the non-illuminated state, the six structures now available define the mechanism of the first phase of the pump at an atomic level, up to the time when recovery of the initial state begins with reprotonation of the retinal Schiff base.

Section snippets

Structural changes in the L state

The L intermediate could be accumulated in a photo-stationary state by illuminating the crystals with a red laser, at either 150 K or 170 K. Figure 1 shows spectra for a single crystal under such conditions. Comparison with difference spectra for authentic L and M at cryogenic temperatures23 indicates that at 150 K about 30% of bacteriorhodopsin is converted to L, and at 170 K about 55% L is produced plus about 10% M. As an approximation, we refined the diffraction data after illumination at 150 K

Data collection

Crystals of wild-type bacteriorhodopsin were grown with the cubic phase method.9 The hexagonal plates were about 80 μ×80 μ×10 μm, with space group P63. The unit cell dimensions were a=b=60.80 Å, c=107.78 Å. After mounting the crystal in a loop and freezing in liquid nitrogen, it was illuminated with a HeNe laser (10 mW, 633 nm) for 60 seconds at 170 K. Diffraction was then measured at 100 K, on beamline 8.2.1 of the Advanced Light Source (ALS, Lawrence-Berkeley Laboratory). The reflections were recorded

Acknowledgements

We thank G. Meigs and G. McDermott of the ALS beamline staff for their essential help, and J. Cupp-Vickery for many valuable suggestions. The work was supported in part by grants to J.K.L. from NIH (R01-GM29498) and from DOE (DEFG03-86ER13525).

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