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 http://resolver.caltech.edu/CaltechAUTHORS:LEEpnas02
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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.
|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, www.rcsb.org (PDB ID code 1MQV).|
|Subject Keywords:||ribonuclease-A, 4-helix bundle, cooperative transitions, nucleation mechanism, microscopic theory, protein molecules, denatured state, landscape, dynamics, pathways|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||22 Nov 2005|
|Last Modified:||26 Dec 2012 08:42|
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