Many membrane proteins show altered activity when their lipid environment is modified, and such changes may well be important in the regulation of membrane protein function in vivo. The major focus of the proposed research will be to determine the effects of specific aspects of the lipid environment, particularly bilayer thickness, on both the structure and the function of bacteriorhodopsin (BR), an integral membrane protein which functions as a light-activated proton pump. In addition, lipid effects on the structure of smaller fragments of BR will be investigated. By examining the effects of lipid bilayer thickness, pH, and other ionic conditions on the structure of these proteins and peptides, the relative roles of hydrophobic and electrostatic forces in determining their structures will be elucidated. In addition, structural studies on the lipids themselves under the same conditions will permit the observation of both sides of the protein-lipid interaction simultaneously. Finally, by correlating the effects of environmental perturbations on the BR structure with the effects on its function, a better understanding of the fundamental mechanism of proton pumping will be gained. The experimental approaches will involve mainly nuclear magnetic resonance (NMR) and optical methods. The structural studies of BR and its fragments will use a novel application of solid state NMR methods, which has recently been developed and tested on BR, to obtain information on the orientation of labeled groups in the protein as it executes rotational diffusion in fluid membrane environments. Both 13C and 2H solid-state NMR studies will be carried out on the protein, and 2H NMR studies will also be performed on the lipids. Infared (IR) and circular dichroism (CD) spectroscopic studies will complement the NMR structural data. The functional studies will involve measurement of proton pumping activity and optical measurements of both steady-state and kinetic spectral properties of BR; the latter will be carried out in collaboration with the University of Virginia, using their flash photolysis apparatus. Finally, x-ray scattering will be used to measure the bilayer thickness for lipids not previously studied.
