A new, state-of-the-art, 500 MHz NMR spectrometer is requested for shared use by a group of six PHS-supported biomedical investigators from the Department of Biochemistry, Tufts University School of Medicine and from the Department of Medicine. The New England Medical Center. This instrument is needed for application in a wide range of biomedical and health related research. Areas of inquiry include: (i) Protease structure, mechanism, inhibitor binding, and inhibitor development. The NMR studies currently focus on a-lytic protease, subtilisin, and the Polovirus 3C protease, but this laboratory is also actively working on IgA proteases and dipeptidyl amino peptidase type IV. (ii) Antibody-DNA interactions. A major goal to determine the structural basis for immune recognition of helical nucleic acid structures. (iii) Vitamin K dependent carboxylation of prothrombin. The 3D solution structures of several prothrombin propeptides are being solved to develop a better understand of the structural elements important in substrate recognition and catalytic carboxylation by the vitamin K- dependent carboxylase. (iv) The structure of the Metal binding sites of prothrombin and the mechanism of the metal dependent interaction of prothrombin with membrane surfaces. This work involves 2D and 3D NMR studies of prothrombin fragment 1. (v) Transforming gene products of the polyoma virus. Preliminary objectives include determining whether structural changes accompany functional mutations in the large T and middle T antigens of polyoma virus. Longer range goals include more detailed structural analysis of large T, and middle T antigens and of functional mutants. (vi) Structure function relationships of the Ras Oncogene Protein. The objectives include understanding the structural basis underlying the functional properties of normal and mutant ras proteins. Five of the six projects listed above involve biological macromolecules with molecular weights at the upper limit of feasibility for NMR structural analysis. However, in most cases, the proteins of interest have been cloned and expressed in quantity. They can be isotopically labeled and subjected to site specific mutagenesis. These properties make them ideal subjects for extending the size boundaries of what can be productively subjected to NMR structural analysis. Nevertheless, the successful pursuit of these projects will require the most recent state- of-the-are instrumentation. The requested instrument is essential for the proposed work and the proposed work will greatly benefit all of the above ongoing PHS-sponsored projects.
