The understanding of the interaction of membranes proteins with membranes is hampered by our lack of physical techniques to characterize the structure of the protein, the effect of the lipid on the protein, and the effect of the protein upon the lipid in its immediate environment. Such techniques can be developed in a reconstituted model system. In such a system it is desirable that the protein adopts the same conformation that it did in the native membrane. Cytochrome b5 is an ideal protein for such a study as it is made on free ribosomes, it is not post-translationally modified and it binds to the membrane via its carboxyl terminus. In addition, the membrane binding domain is relatively small and it contains tryptophans which are fluorescent. The structure of the hydrophobic membrane-binding domain can be investigated by fluorescence quenching by insertion into vesicles made from a series of phospholipids which are brominated at different positions in the acyl chain. The efficiency of quenching gives a measure of the depth, in the membrane, of the fluorescent tryptophans. The quenching in vesicles made from mixtures of brominated and non-brominated lipids indicates the composition of the """"""""boundary lipid"""""""" and the accessibility of the tryptophans to these collisional quenchers. The acyl chains and the polar head groups of the lipids will be systematically varied to determine the role these domains play in determining the protein, and lipid, structure; alternation of the structure of either would modify the quenching. The structure, orientation, and motions of the hydrophobic domain will be influenced by its primary sequence and by the hydrophobic domain. To examine these possibilities protein analogues will be generated by site specific mutagenesis, by chemical synthesis or by enzymic or chemical modification of the cytochrome. CD measurements will give information on secondary structure, NMR on structure and dynamics of proteins and lipids, and calorimetry on lipid structure and the enthalpy of binding. Animal membranes contain over 100 different lipid species. This project will determine the role these lipids play in protein dynamics and topography and the effect of proteins on lipid dynamics and topography.
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