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Structural features of cytochrome c' folding intermediates revealed by fluorescence energy-transfer kinetics

Lee, Jennifer C. and Engman, K. Cecilia and Tezcan, F. Akif and Gray, Harry B. and Winkler, Jay R. (2002) Structural features of cytochrome c' folding intermediates revealed by fluorescence energy-transfer kinetics. Proceedings of the National Academy of Sciences of the United States of America, 99 (23). pp. 14778-14782. ISSN 0027-8424.

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We employed fluorescence energy-transfer probes to investigate the polypeptide dynamics accompanying cytochrome c' folding. Analysis of fluorescence energy-transfer kinetics from wild-type Trp-72 or Trp-32 in a crystallographically characterized (1.78 Angstrom) Q1A/F32W/W72F mutant shows that there is structural heterogeneity in denatured cytochrome c'. Even at guanidine hydrochloride concentrations well beyond the unfolding transition, a substantial fraction of the polypeptides (approximate to 50%) adopts compact conformations (tryptophan-to-heme distance, approximate to 25 Angstrom) in both pseudo-wild-type (Q1A) and mutant proteins. A burst phase (less than or equal to 5 ms) is revealed when stopped flow-triggered refolding is probed by tryptophan intensity: measurements on the Q1A protein show that approximate to 75% of the Trp-72 fluorescence (83% for Trp-32) is quenched within the mixing deadtime, suggesting that most of the polypeptides have collapsed.

Item Type:Article
Additional Information:Copyright © 2002 by the National Academy of Sciences. Contributed by Harry B. Gray, September 22, 2002. Published online before print October 29, 2002, 10.1073/pnas.192574099 This work was supported by National Science Foundation Grants MCB-9974477 and DBI-9876443 and the Arnold and Mabel Beckman Foundation. J.C.L. acknowledges the Parsons Foundation for a graduate fellowship, and K.C.E. acknowledges the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) for a fellowship. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, (PDB ID code 1MQV).
Subject Keywords:ribonuclease-A, 4-helix bundle, cooperative transitions, nucleation mechanism, microscopic theory, protein molecules, denatured state, landscape, dynamics, pathways
Record Number:CaltechAUTHORS:LEEpnas02
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:988
Deposited By: Tony Diaz
Deposited On:22 Nov 2005
Last Modified:14 Nov 2014 19:18

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