STRUCTURE AND INTERACTION OF PROTEINS WITH MEMBRANES

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We are studying the structure of proteins which interact with the eukaryotic cell surface, and the mechanism by which proteins are internalized by cells. Wheat germ agglutinin (WGA), a plant lectin which binds to cell surface carbohydrates, is being studied by x-ray crystallography and chemical methods. Studies of the mechanism of internalization of extracellular proteins and the effects of chemical modification of cytotoxicity are being done on diphtheria toxin (DT). Based on the recently determined amino acid sequence and additional high resolution x-ray data for WGA 2, a reconstructed and refined model of the WGA structure will be used for analysis of subunit interactions and stereochemistry of saccharide binding to the protein. The amino acid sequence of WGA isolectin 1 will be determined, and by model building, those functional groups which determine the differential saccharide affinities of these two isolectins determined. Finally, the crystal structure of a complex of WGA 2 with a glycopeptide from the major red blood cell membrane protein, glycophorin, will be determined crystallographically. The structure of this complex will be a more definitive model for protein-cell surface saccharide interactions than the smaller saccharide complexes. The nature of the interaction of DT with cell surface groups of several cell lines in culture, and the requirements for internalization of the toxic A-chain of DT will be studied by chemical methods. The cell surface molecules to which DT binds will be determined by chemical modification studies, and through the use of specific inhibitors of cellular biosynthetic processes. Chemically modified DT will be used to characterize the membrane translocation process, and to isolate the membrane-bound molecules which carry out the transport. Similarly modified proinsulin or other hormones will be tested to determine whether the same mechanism is utilized in their internalization. These studies bear on the question of membrane structure and transport. The structures of membrance surface molecules are influenced by factors such as malignant transformation, cellular millieu and cell density, and control adhesion and assembly processes. Cellular membrane transport determines the internal state of the cell, and is linked to cell surface structure in the effects of hormones, viruses, antibodies and toxins upon binding to receptors. It may also find application in the intracellular delivery of therapeutic agents.