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The folding kinetics of the SDS-induced molten globule form of reduced cytochrome c.
Biochemistry. 2008 May 13; 47(19):5450-9.B

Abstract

The folding of reduced cytochrome c (redcyt c) is increasingly being recognized as undergoing a mechanism that deviates from a two-state process. In previous far-UV TRORD studies of redcyt c folding, a rapidly forming intermediate was attributed to the appearance of a molten-globule-like (MG) state [Chen, E., Goldbeck, R. A., and Kliger, D. S. (2003) J. Phys. Chem. A 107, 8149-8155]. A slow folding phase (>1 ms) was identified with the formation of native (N) secondary structure from that MG form. Here, using 0.65 mM SDS to induce the MG state in oxidized cytochrome c, folding of redcyt c was triggered with fast photoreduction and probed from early microseconds to milliseconds using far-UV TRORD spectroscopy. The kinetics of the reaction are described with a time constant of 50 +/- 16 ms, which corresponds to 1 +/- 0.6 ms upon extrapolation of the data to zero SDS concentration. The latter folding time is about 5 times faster than the calculated GuHCl-free time constant of 5.5 +/- 1.4 ms for slow-phase folding obtained in our previous study. This ratio of rates would be consistent with a scenario in which 20-30% MG that is suggested to form in the fast phase of redcyt c folding in GuHCl is an obligatory intermediate. The native state forms from this obligatory intermediate with an observed rate, k(f) = fk(G-->N) where f is the fractional population of MG and k(G-->N) is the microscopic rate for MG --> N. Calculation and comparison of the m(#)/m values show agreement within the uncertainties between the SDS (approximately 0.5) and GuHCl (approximately 0.3) based redcyt c folding experiments, suggesting that the two experiments report on comparable intermediates. The m values were obtained from far-UV CD SDS titration experiments, from which calculated thermodynamic parameters allowed estimation of the reduction potential for the MG state to be approximately 155 mV (-15 kJ/mol) vs NHE which, like the reduction potential for the native state, is more favorable than that for the unfolded protein.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, UniVersity of California, Santa Cruz, California 95064, USA. chen@chemistry.ucsc.eduNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

18416561

Citation

Chen, Eefei, et al. "The Folding Kinetics of the SDS-induced Molten Globule Form of Reduced Cytochrome C." Biochemistry, vol. 47, no. 19, 2008, pp. 5450-9.
Chen E, Van Vranken V, Kliger DS. The folding kinetics of the SDS-induced molten globule form of reduced cytochrome c. Biochemistry. 2008;47(19):5450-9.
Chen, E., Van Vranken, V., & Kliger, D. S. (2008). The folding kinetics of the SDS-induced molten globule form of reduced cytochrome c. Biochemistry, 47(19), 5450-9. https://doi.org/10.1021/bi702452u
Chen E, Van Vranken V, Kliger DS. The Folding Kinetics of the SDS-induced Molten Globule Form of Reduced Cytochrome C. Biochemistry. 2008 May 13;47(19):5450-9. PubMed PMID: 18416561.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The folding kinetics of the SDS-induced molten globule form of reduced cytochrome c. AU - Chen,Eefei, AU - Van Vranken,Vanessa, AU - Kliger,David S, Y1 - 2008/04/17/ PY - 2008/4/18/pubmed PY - 2008/6/11/medline PY - 2008/4/18/entrez SP - 5450 EP - 9 JF - Biochemistry JO - Biochemistry VL - 47 IS - 19 N2 - The folding of reduced cytochrome c (redcyt c) is increasingly being recognized as undergoing a mechanism that deviates from a two-state process. In previous far-UV TRORD studies of redcyt c folding, a rapidly forming intermediate was attributed to the appearance of a molten-globule-like (MG) state [Chen, E., Goldbeck, R. A., and Kliger, D. S. (2003) J. Phys. Chem. A 107, 8149-8155]. A slow folding phase (>1 ms) was identified with the formation of native (N) secondary structure from that MG form. Here, using 0.65 mM SDS to induce the MG state in oxidized cytochrome c, folding of redcyt c was triggered with fast photoreduction and probed from early microseconds to milliseconds using far-UV TRORD spectroscopy. The kinetics of the reaction are described with a time constant of 50 +/- 16 ms, which corresponds to 1 +/- 0.6 ms upon extrapolation of the data to zero SDS concentration. The latter folding time is about 5 times faster than the calculated GuHCl-free time constant of 5.5 +/- 1.4 ms for slow-phase folding obtained in our previous study. This ratio of rates would be consistent with a scenario in which 20-30% MG that is suggested to form in the fast phase of redcyt c folding in GuHCl is an obligatory intermediate. The native state forms from this obligatory intermediate with an observed rate, k(f) = fk(G-->N) where f is the fractional population of MG and k(G-->N) is the microscopic rate for MG --> N. Calculation and comparison of the m(#)/m values show agreement within the uncertainties between the SDS (approximately 0.5) and GuHCl (approximately 0.3) based redcyt c folding experiments, suggesting that the two experiments report on comparable intermediates. The m values were obtained from far-UV CD SDS titration experiments, from which calculated thermodynamic parameters allowed estimation of the reduction potential for the MG state to be approximately 155 mV (-15 kJ/mol) vs NHE which, like the reduction potential for the native state, is more favorable than that for the unfolded protein. SN - 1520-4995 UR - https://www.unboundmedicine.com/medline/citation/18416561/The_folding_kinetics_of_the_SDS_induced_molten_globule_form_of_reduced_cytochrome_c_ DB - PRIME DP - Unbound Medicine ER -