This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PROJECT VI: Polenova - Project will investigate structure and conformational dynamics of a 68 kDa- vanadium chloroperoxidase in the solid state using multidimensional NMR spectroscopy. The focus on two major objectives: 1) determine the tertiary structure of chloroperoxidase using solid-state NMR data only;2) to establish the multiple timescale conformational dynamics in the protein. To accomplish these objectives, three specific aims will be pursued: 1) 13C and 15N resonance assignments of uniformly labeled vanadium chloroperoxidase;2) determination of long-range tertiary constraints and structure calculation;3) investigation of multiple timescale conformational dynamics in uniformly and sparsely isotopically enriched protein. These studies would on one hand help elucidate the role of internal conformational dynamics in the substrate specificity of the vanadium haloperoxidases, and on the other, expand the capabilities of solids NMR to structural characterization of large noncrystalline proteins currently intractable by X-ray crystallography or solution NMR. Experimental plan. We completed the specific aim 1 and made significant progress for aim 2. The following experiments were conducted first: 1) heteronuclear 13C-15N two-dimensional correlation experiments to establish backbone resonance assignments, using the double cross polarization (DCP) mixing scheme;1 2) homonuclear two-dimensional 13C-13C correlation experiments to establish the sidechain assignments, using RFDR2 and RAD3 mixing schemes. In the possible case of the insufficient spectral resolution, the three-dimensional NCC protocols were pursued, involving NCACO and NCACB band-selective magnetization transfer schemes. With these experiments in hand, we have to assigned most of the resonances in the protein.
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