This proposal focuses on several important issues underlying the folding, stability, dynamics and functional state dependence of prosthetic group binding proteins. Our goal is to contribute to the understanding of the contribution of structure and dynamics to the functional competence of flavin and heme proteins, such as those involved in electron transfer. Electron transfer reactions between proteins control the generation, flow and use of energy in biological systems and one third of known enzymes are involved in oxidation and reduction. Accordingly, redox dysfunctional is directly involved with a variety of fetal or debilitating syndromes. We are most interested in exploring why many b-heme and flavin binding proteins display considerate structure and stability in their corresponding apostates and how binding modifies both the prosthetic group and the protein. Specifically, we propose to characterize the structural cooperativity of three apoproteins using hydrogen exchange, NMR relaxation, and a variety of structural NMR methods; we propose to directly quantify the redox- state dependence of dynamics of several holoproteins in an effort to understand the nature of the prosthetic group-protein interface; we propose to use our accumulated knowledge to continue the design and engineering of a series of minimalist proteins, """"""""maquettes"""""""", with the ultimate aim of constructing functionally useful and catalytically active mini-proteins which might serve as advanced pharmaceutics and (bio)chemical reagents; and we propose to characterize the complex between cytochrome c and its binding domain in Apaf-1, an interaction that is a key event in apoptosis, programmed cell death. Each of these specific aims will require the extensive use of multi-nuclear and multi- dimensional NMR spectroscopy and will take advantage o f recent methodological developments and novel capabilities, particularly NMR relaxation methods and a high pressure NMR capability.
| Caro, José A; Wand, A Joshua (2018) Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology. Methods 148:67-80 |
| Liu, Weixia; Rumbley, Jon N; Englander, S Walter et al. (2009) Fast structural dynamics in reduced and oxidized cytochrome c. Protein Sci 18:670-4 |
| Gledhill Jr, John M; Joshua Wand, A (2008) Optimized angle selection for radial sampled NMR experiments. J Magn Reson 195:169-78 |
| Bedard, Sabrina; Mayne, Leland C; Peterson, Ronald W et al. (2008) The foldon substructure of staphylococcal nuclease. J Mol Biol 376:1142-54 |
| Calhoun, Jennifer R; Liu, Weixia; Spiegel, Katrin et al. (2008) Solution NMR structure of a designed metalloprotein and complementary molecular dynamics refinement. Structure 16:210-5 |
| Gledhill, John M; Wand, A Joshua (2007) Phasing arbitrarily sampled multidimensional NMR data. J Magn Reson 187:363-70 |
| Valentine, Kathleen G; Pometun, Maxim S; Kielec, Joseph M et al. (2006) Magnetic susceptibility-induced alignment of proteins in reverse micelles. J Am Chem Soc 128:15930-1 |
| Pometun, Maxim S; Peterson, Ronald W; Babu, Charles R et al. (2006) Cold denaturation of encapsulated ubiquitin. J Am Chem Soc 128:10652-3 |
| Koder, Ronald L; Valentine, Kathleen G; Cerda, Jose et al. (2006) Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions. J Am Chem Soc 128:14450-1 |
| Whitten, Steven T; Kurtz, Andrew J; Pometun, Maxim S et al. (2006) Revealing the nature of the native state ensemble through cold denaturation. Biochemistry 45:10163-74 |
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