This application requests continued support for an ongoing program of study on the physicochemical and biological properties of proteins and peptides in solution with emphasis on their interaction with each other and with small molecules. Both experimental and theoretical research projects are proposed. The two experimental projects are: (1) A combined CD-NMR study of the solution conformation of the Bradykinin-like agonists and antagonists: (D-Phe7)-, (Thi5,8)-, and (Thi5,8,D-Phe7)-bradykinin, where Thi is Beta-2-thienylalanine. The first analog is an agonist or an antagonist depending on the tissue; the second is a super agonist; and the third is a good antagonist. Elucidation and comparison of their solution conformation is essential for eventual understanding of their biological activities. (2) A study of the binding of subdomains of the 4 kDa C-terminal moiety of tubulin by MAP-2. Peptides corresponding to short discrete aminoacid sequences within the Alpha- and Beta-chain of the C-terminal moiety will be synthesized by the solid-phase method of Merrifield. The binding of each of these peptides to MAP-2 will be quantitated by sedimentation analysis in the Airfuge using radiolabeled peptide as a tracer. It is anticipated that the results may indicate which sequence might confer preferential binding of the Alpha-chain of tubulin to MAP-2. Our theoretical calculations on the mass transport of interacting macromolecules will be continued and extended to include: (1) The broad-zone Sephadex filtration of the myoglobin-ovalbumin system in order to answer certain questions raised by published experimental results on this model, complexing system; (2) The small-zone gel chromatography of systems in which the binding of a peptide (or other ligand) promoters dimerization of a protein, in order to provide the basis for estimating binding constants in systems such as MAP2-4 kDa C-terminal peptide fragment of tubulin; and (3) zone electrophoresis of hybridizing enzymes with kinetic control.
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