We are interested in the structure and dynamics of membrane components, particularly in relation to membrane assembly and function. Electron spin resonance spectroscopy (ESR) is commonly used to examine the dynamics of membrane lipids but our preliminary data with a spin-labeled protein indicate that ESR may be a valuable approach for studying the interaction of proteins with membranes. We intend to test this potentially useful application of ESR technology. Our studies with photoreactive derivatives of stearoylglucosamine-l-(14C) have shown that the sequenced coat protein of coliphage M13 when oriented across artificial vesicles provides a perfect """"""""ruler"""""""" for determining the zone of reactivity of a given probe with vicinal molecules. Once defined, such probes can be used to pinpoint the location of other proteins (e.g., Braun's lipoprotein) with respect to the bilayer, to identify newly inserted membrane components, and to establish the kinetics with which individual segments or subunits or proteins insert into membranes, for example, during membrane assembly, cholera toxin attack, or complement-mediated lysis. We plant to synthesize and characterize photoreactive lipid derivatives which vary in composition and charge and in placement of the photolabile moiety. An important objective is to correlate the kinetics of physical insertion as revealed by ESR or photolabeling studies with functional insertion as revealed by biochemical assays or permeability studies. We also want to develop a model system to study the mechanism of membrane fusion as promoted by the extracted glycoproteins of Sendai virus since we have shown that they do not have to be embedded in a lipid matrix to function. Our immediate goals are to define 1) the kinetics with which specific proteins insert into membranes and 2) the functional destination of inserted proteins. Our long-term goals are to define 1) the fate of protein """"""""leader"""""""" sequences and 2) the effect of lipid structure on the course of protein insertion. Knowing how the lytic complex of complement works or how cholera toxin attacks a cell or how two membranes fuse during endocytosis, secretion and infection by certain lipid-enveloped viruses will have an immediate impact on the medical field.
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