EPR-Detected Folding Kinetics of Externally Located Cysteine-Directed Spin-Labeled Mutants of Iso-1-cytochrome c
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- 作者：Kim DeWeerd ; Vladimir Grigoryants ; Yuhua Sun ; Jacquelyn S. Fetrow ; and Charles P. Scholes
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：December 25, 2001
- 年：2001
- 卷：40
- 期：51
- 页码：15846 - 15855
- 全文大小：118K
- 年卷期：v.40,no.51(December 25, 2001)
- ISSN：1520-4995
文摘
We report the application of our newly developed dielectric resonator-based flow and stopped-flow kinetic EPR systematically to probe protein folding in yeast iso-1-cytochrome c at cysteine-directedspin-labeled locations. The locations studied have not been previously directly probed by other techniques,and we observe them on a time scale stretching from 50 
s/entities/mgr.gif">s to seconds. On the basis of crystal structureand homology information, the following mutation-tolerant, externally located cysteine labeling sites werechosen (in helices, T8C, E66C, and N92C; in loops, E21C, V28C, H39C, D50C, and K79C), and labelingat these sites was not destabilizing. Dilution of denaturant was used to induce folding and thereby tocause a change in the spin label EPR signal as folding altered the motion of the spin label. Under foldingconditions, including the presence of imidazole to eliminate kinetic trapping due to heme misligation, aphase of folding on the 20-30 ms time scale was found. This phase occurred not only at the T8C andN92C labeling sites in the N- and C-terminal helices, where such a phase has been associated with foldingin these helices, but overall at labeling sites throughout the protein. In the absence of imidazole the 20-30 ms phase disappeared, and another phase having the time scale of 1 s appeared throughout the protein.There was evidence under all conditions for a burst phase on a scale of less than several millisecondswhich occurred at labeling positions V28C, H39C, D50C, E66C, and K79C in the middle of the proteinsequence. At spin-labeled D50C rapid-mix flow EPR indicated a very short ~50 s/entities/mgr.gif">s phase possiblyassociated with the prefolding or compaction of the loop to which D50 belongs. Spin labels have beencriticized as perturbing the phenomena which they measure, but our spin labeling strategy has reportedcommon kinetic themes and not perturbed, disconnected kinetic events.