Shahab Rouhani

Structure of an Early Intermediate in the M-State Phase of the Bacteriorhodopsin Photocycle

The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.

Characterization of Conditions Required for X-Ray Diffraction Experiments with Protein Microcrystals

The x-ray exposure at which significant radiation damage occurs has been quantified for frozen crystals of bacteriorhodopsin. The maximum exposure to approximately 11-keV x-rays that can be tolerated for high-resolution diffraction experiments is found to be approximately 10(10) photons/microm(2), very close to the value predicted from limits that were measured earlier for electron diffraction exposures. Sample heating, which would further reduce the x-ray exposure that could be tolerated, is not expected to be significant unless the x-ray flux density is well above 10(9) photons/s-microm(2). Crystals of bacteriorhodopsin that contain approximately 10(11) unit cells are found to be large enough to give approximately 100 high-resolution diffraction patterns, each covering one degree of rotation. These measurements are used to develop simple rules of thumb for the minimum crystal size that can be used to record x-ray diffraction data from protein microcrystals. For work with very small microcrystals to be realized in practice, however, it is desirable that there be a significant reduction in the level of background scattering. Background reduction can readily be achieved by improved microcollimation of the x-ray beam, and additional gains can be realized by the use of helium rather than nitrogen in the cold gas stream that is used to keep the protein crystals frozen.

Crystal Structure of the Bromide-Bound D85S Mutant of Bacteriorhodopsin: Principles of Ion Pumping

We report the crystal structure of a bromide-bound form of the D85S mutant of bacteriorhodopsin, bR(D85S), a protein that uses light energy rather than ATP to pump halide ions across the cell membrane. Comparison of the structure of the halide-bound and halide-free states reveals that both displacements of individual side-chain positions and concerted helical movements occur on the extracellular side of the protein. Analysis of these structural changes reveals how this ion pump first facilitates ion uptake deep within the cell membrane and then prevents the backward escape of ions later in the pumping cycle. Together with the information provided by structures of intermediate states in the bacteriorhodopsin photocycle, this study also suggests the overall design principles that are necessary for ion pumping.

Crystal structures of bR(D85S) favor a model of bacteriorhodopsin as a hydroxyl-ion pump

Structural features on the extracellular side of the D85S mutant of bacteriorhodopsin (bR) suggest that wild‐type bR could be a hydroxyl‐ion pump. A position between the protonated Schiff base and residue 85 serves as an anion‐binding site in the mutant protein, and hydroxyl ions should have access to this site during the O‐intermediate of the wild‐type bR photocycle. The guanidinium group of R82 is proposed (1) to serve as a shuttle that eliminates the Born energy penalty for entry of an anion into this binding pocket, and conversely, (2) to block the exit of a proton or a related proton carrier.

UV microscopy at 280 nm is effective in screening for the growth of protein microcrystals

This paper describes the relatively simple modification of a light microscope to operate with ultraviolet-based optics. Using a 280 nm UV illumination source, protein microcrystals are readily visualized in gels made from hydrated bilayers of phospholipids. Non-colored proteins stand out as clearly as colored proteins in this system, the imaging of which is based on UV absorption by tryptophan residues. In addition, protein crystals are easily distinguished from salt crystals. Artifacts from the lipid-based crystallization medium, which are frequently seen in brightfield microscopy, are greatly reduced when viewed in this UV-based microscope.

Microcrystal screening with a novel design for beamline-mountable crystallization wells

A simple and flexible system is described for in situ screening of microcrystals of membrane proteins that are grown within a connected-bilayer matrix formed by hydrated lipids. Using sheets of appropriate polymer materials to create a thin multiwell cassette, crystals can be evaluated by UV microscopy as well as by more conventional forms of light microscopy. Crystallization wells can be individually excised and mounted for diffraction screening on a synchrotron X-ray source. In addition, crystallization hit rates were significantly improved by employing a vapor diffusion approach rather than the batch crystallization method that is normally used with hydrated-lipid gels.