UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

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The major objective of this proposal is to develop methods that will elucidate the dynamic structure or peptides and proteins in both solution and the solid state. 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 2H-,13C-, and 15N-labeled amino acids and incorporate them into Staphylococcal Nuclease and, perhaps, other proteins. These molecules will then be studied by high field (200-500 MHz) NMR. In one project, (15N, 1-13C)-leucine will be prepared and incorporated in Nuclease to study the solution environment of the active site. In a second project, Nuclease containing a variety of synthetic C-13 methyl-labeled amino acids will be prepared and magic angle spinning techniques will be used to obtain a more detailed picture- of the region around the various labeled methyl groups in the solid state. Finally, in the third project, (d6)-proline will be employed in an attempt to extend a new method that we have developed for assigning cis signals in the proton spectra of peptides. To implement these projects, I am requesting support for three undergraduate students for five years. They will bear primary responsibility for the synthesis of all the labeled amino acids and peptides and they will routinely purify and analyze their products by chromatography and NMR. Also, York's recent acquisition of a 200 MHz broadband FT-NMR (with the aid of an MBRS supplemental instrumentation award) will enable these students to participate fully in characterizing their C-13, N-15, and deuterium-labeled products and in gathering and interpreting relaxation time, Nuclear Overhauser Effect, and exchange data using sophisticated pulse techniques not generally available to undergraduates.