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

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 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.

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