Variable velocity liquid flow EPR applied to submillisecond protein folding.
We have developed a variable velocity, rapid-mix, continuous-flow method for observing and delineating kinetics by dielectric resonator-based electron paramagnetic resonance (EPR). The technology opens a new facet for kinetic study of radicals in liquid at submillisecond time resolution. The EPR system (after Sienkiewicz, A., K. Qu, and C. P. Scholes. 1994. Rev. Sci. Instrum. 65:68-74) accommodated a miniature quartz capillary mixer with an approximately 0.5 microliter delivery volume to the midpoint of the EPR-active zone. The flow velocity was varied in a preprogrammed manner, giving a minimum delivery time of approximately 150 microseconds. The mixing was efficient, and we constructed kinetics in the 0.15-2. 1-ms time range by plotting the continuous wave EPR signal taken during flow versus the reciprocal of flow velocity. We followed the refolding kinetics of iso-1-cytochrome c spin-labeled at Cysteine 102. At 20 degrees C, upon dilution of guanidinium hydrochloride denaturant, a fast phase of refolding was resolved with an exponential time constant of 0.12 ms, which was consistent with the "burst" phase observed by optically detected flow techniques. At 7 degrees C the kinetic refolding time of this phase increased to 0.5 ms.
Grigoryants
V M
VM
Department of Chemistry, University at Albany, State University of New York, Albany, New York 12222, USA.
Veselov
A V
AV
Scholes
C P
CP
D055592:Biophysical Phenomena; D001703:Biophysics; D003497:Cyclic N-Oxides; D003545:Cysteine / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry; D045304:Cytochromes c; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D008698:Mesylates; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels
C014059:CYC1 protein, S cerevisiae; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D008698:Mesylates; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate; D045304:Cytochromes c; D003545:Cysteine