The main objective of this research is to elucidate and understand the details of protein-oligosaccharide interaction at the molecular level. In their interactions with carbohydrates, lectins serve as models for studying the factors that control the specificity and binding energetics between proteins and complex carbohydrates. The determination of the three-dimensional structures of these lectins and their complexes are an essential component in understanding the molecular details of the recognition process. The specific interaction of proteins and carbohydrates is of critical importance in cell-cell adhesion, cell-virus recognition, cell-protein recognition, and other cellular regulation processes. The molecular mechanism involved is fundamental to most viral infections. The information that is currently available concerning simple monosaccharide-protein interaction, will be extended by lectin-oligosaccharide and lectin-Asn-linked-oligosaccharides structural investigations. This will be accomplished by an experimental program that determines the molecular structure by the method of x-ray crystallography. The structure of the agglutinin isolated from pisum sativum (PSA), a 49K dalton lectin, has been determined to 1.9 A resolution by conventional crystallographic methods. The gene coding for PSA has been expressed in E.coli and crystals of the single chain recombinant protein have been obtained that diffract to high resolution. The structure of this recombinant molecule will be determined by single crystal x-ray diffraction using molecular replacement and/or multiple isomorphous replacement methods. Site specific mutants of the recombinant PSA have been expressed and will be crystallized both alone and complexed with Asn-linked- oligosaccharides. Efforts to explore the tolerable alterations in the amino acid sequence of recombinant PSA and their effect on carbohydrate binding and specificity will be accomplished. Crystals, suitable for diffraction studies, of several other lectins have been prepared.
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