Much of the biological activity of the cell is organized at membrane surfaces; by proteins that are integral membrane proteins, by proteins that are loosely associated with the membrane surface, and by proteins that are transiently anchored by elements containing fatty acyl chains. Yet, there is little structural information on which to base a physical understanding of this organization. Application of traditional approaches to structure elucidation, including high resolution nuclear magnetic resonance (NMR) and X-ray crystallography, are limited when systems are assembled on partially ordered arrays such as lipid bilayers. This proposal continues the development of special NMR based techniques that are applicable to ordered arrays of lipid bilayers that can serve as model systems for the structural study of membrane associated proteins. The NMR methods use orientational constraints derived from anisotropic interactions such as residual dipolar coupling and chemical shift anisotropy. These interactions are measured in arrays of lipid bilayer discs (bicelles) that undergo magnetic field induced cooperative ordering. The bicelle systems will be optimized to contain lipid constituents deemed necessary for proper representation of membrane protein interaction, and the NMR methodology will be modified to allow simultaneous measurement of many anisotropic interactions in protein systems isotopically labeled with NMR active nuclei. A specific application target will be used to direct the course of method development. This target is ADP ribosylation factor (ARF1), a protein important in protein traffic particularly in and around the Golgi. ARF1 is a 20 kDa, N-myristoylated GTPase in which activation (GTP binding) is tightly coupled to membrane binding. The structural basis of this essential, reversible membrane association will be investigated. There is, perhaps, no better example of the complexity of organization of cellular chemistry than in protein traffic through the membranes of the Golgi. Elucidation of molecular details of ARF's role in this process can be a stepping stone to understanding a variety of membrane centered processes.
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