UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

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The major objective of this proposal is to develop and apply methods to elucidate the dynamic structure of proteins in solution. It is now generally recognized that proteins do not have static structures and that X-ray data must be supplemented by motional information about sidechain and backbone atoms if a more complete understanding of the nature of protein interactions is to be achieved. We propose to synthesize a variety of 13C- and 15N-labeled amino acids and incorporate them into staphylococcal nuclease, despentapeptideinsulin amide and the E. coli signal transducing protein, IIIGlc. These molecules will then be studied by high field (200-600 MHz) nuclear magnetic resonance (nmr) spectroscopy. We are especially interested in studying sidechain motion in these proteins and therefore our primary purpose is to synthesize amino acids labeled at every other sidechain carbon with C-13. Then with site-specific T1, T2, and long-range NOE data in hand, we hope to be able to paint a detailed picture of the structure and dynamics at and around these labeled sites. To implement these projects, I am requesting support for three undergraduate students for four years. They will bear primary responsibility for the synthesis of all the labeled amino acids, peptides and insulin derivatives and they will routinely purify and analyze their products by chromatography and nmr. York's 200 MHz broadband FT-nmr (acquired with the aid of an MBRS supplemental instrumentation award) and new UNIX workstation and our recently purchased solid phase peptide synthesizer will enable these students to participate fully in preparing, characterizing, and analyzing their C-13 and N-15 labeled products with the aid of forefront technology not generally available to undergraduates.