Utilizing a combination of experimental nmr and molecular dynamics simulation methods, my research is currently the investigation of phosphorylation-mediated protein-protein interactions to understand molecular recognition and functional properties of tyrosine phosphorylation. The general direction of this research is leading to investigations of biophysical properties of other integral membrane proteins. An award would significantly diminish the obligations 1 have within the Department by providing the means to hire personnel to assume teaching and undergraduate computer-instruction obligations. The increase in time available for research will greatly enhance my research program particularly considering the scope of both experimental nmr and computational methods we employ. The Dept. of Medicinal Chemistry is committed to a strong research program. Between collaborators within. Medicinal Chemistry and the Structure Group in Biological Sciences, including five faculty in protein crystallography, the environment is excellent to carry out my research goals. Mediation of protein-protein interactions through tyrosine phosphorylation is a fundamental feature of many vital signal transduction pathways in cells. This proposal describes nmr studies, assisted by computational approaches, to determine 3-dimensional structure at the tyrosyl binding site and contact region of certain protein complexes. The specific complexes addressed here are erythrocyte band 3 plus the glycolytic enzymes, aldolase or glyceraldehyde-3-phosphate dehydrogenase, and Ig- alpha, one component of the B-cell antigen receptor (BCR) complex, plus Lyn kinase. Association of band 3 with glycolytic enzymes leads to reversible inactivation of the enzymes, and phosphorylation of an N- terminal tyrosine of band 3 by p72syk in erythrocyte prevents association. How phosphorylation mediates this association and the mechanism of band 3 inhibition are unknown. Using a peptide derived from the N-terminal region and two-dimensional exchange-transferred NOE spectroscopy, structural information will be obtained on the bound band 3 peptide that can provide an understanding of the mediation of protein-protein association by tyrosine phosphorylation. With regard to Lyn, a src-family tyrosine kinase, Lyn is thought to phosphorylate the ARH- l motif of Ig-alpha in an initial signaling event stimulated by antigen binding to B-cells. Again, using a peptide (from ARH-1) and transferred NOESY, the conformation of peptide bound in the active site of Lyn will be determined. The results of these studies should not only increase the understanding of kinase substrate recognition, but also aid in the design of inhibitors of Lyn and other tyrosine kinases involved in cell growth regulation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Scientist Development Award - Research (K02)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Purdue University
Schools of Pharmacy
West Lafayette
United States
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