The purpose of this work is to exploit our one-of-a-kind, high sensitivity flow and stopped-flow EPR to probe the real time folding and recognition of spin labeled biomolecules with a time resolution extending from 50 microseconds to seconds. The areas to be investigated are: Iso-1-Cytochrome c. This work builds on our high yield expression system for externally located, nonperturbing, cysteine-directed mutants of iso-1-cytochrome c. With single mutants, we will measure the location, time scale, and activation energy of rapid, submillisecond folding. With bi-labeled mutants and rapid-mix flow EPR we will measure the time development of distant tertiary structure and helices. RNA-Protein Folding/Recognition. We will study folding and recognition of a stem-loop RNA-HIV-1 nucleocapsid protein complex. Using a specific retroviral stem loop RNA spin labeled at its 5' terminal and spin labeled HIV NCP7 protein, we will measure the time course for structural changes leading to the final RNA-protein complex. DnaK, a Molecular Chaperone. We will probe recognition of a spin-labeled hydrophobic peptide by the heat shock protein DnaK and adaptation of this peptide binding in response to ATP-induced conformational change of DnaK. T4 Lysozyme. Using rapid-mix submillisecond flow EPR, we will study bi-labeled T4 lysozyme to find the time scale for protein helix formation and to find the time scale for residues far apart in sequence to reach their nearby folded conformation. Technical Development. We will improve our dielectric resonator based technology to achieve optimum coordination of flow and field sweep, minimal use of reagents, and even shorter dead times.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biophysical Chemistry Study Section (BBCB)
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Basavappa, Ravi
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State University of New York at Albany
Schools of Arts and Sciences
United States
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Sun, Yan; Borbat, Peter P; Grigoryants, Vladimir M et al. (2015) Pulse dipolar ESR of doubly labeled mini TAR DNA and its annealing to mini TAR RNA. Biophys J 108:893-902
Sun, Yan; Zhang, Ziwei; Grigoryants, Vladimir M et al. (2012) The internal dynamics of mini c TAR DNA probed by electron paramagnetic resonance of nitroxide spin-labels at the lower stem, the loop, and the bulge. Biochemistry 51:8530-41
McConnell, Iain L; Grigoryants, Vladimir M; Scholes, Charles P et al. (2012) EPR-ENDOR characterization of (17O, 1H, 2H) water in manganese catalase and its relevance to the oxygen-evolving complex of photosystem II. J Am Chem Soc 134:1504-12
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Zhang, Zhiwen; Xi, Xiangmei; Scholes, Charles P et al. (2008) Rotational dynamics of HIV-1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis. Biopolymers 89:1125-35