Structural features of cytochrome c ′ folding intermediates revealed by fluorescence energy-transfer kinetics
- 29 October 2002
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 99 (23) , 14778-14782
- https://doi.org/10.1073/pnas.192574099
Abstract
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 Å) 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 (≈50%) adopts compact conformations (tryptophan-to-heme distance, ≈25 Å) in both pseudo-wild-type (Q1A) and mutant proteins. A burst phase (≤5 ms) is revealed when stopped flow-triggered refolding is probed by tryptophan intensity: measurements on the Q1A protein show that ≈75% of the Trp-72 fluorescence (83% for Trp-32) is quenched within the mixing deadtime, suggesting that most of the polypeptides have collapsed.Keywords
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