title: 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 A) 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 ( approximately 50%) adopts compact conformations (tryptophan-to-heme distance, approximately 25 A) in both pseudo-wild-type (Q1A) and mutant proteins. A burst phase (< or =5 ms) is revealed when stopped flow-triggered refolding is probed by tryptophan intensity: measurements on the Q1A protein show that approximately 75% of the Trp-72 fluorescence (83% for Trp-32) is quenched within the mixing deadtime, suggesting that most of the polypeptides have collapsed.
authors.affiliation: Beckman Institute, California Institute of Technology, Pasadena 91125, USA.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Lee
authors.forename: K Cecilia
authors.initials: KC
authors.lastname: Engman
authors.forename: F Akif
authors.initials: FA
authors.lastname: Tezcan
authors.forename: Harry B
authors.initials: HB
authors.lastname: Gray
authors.forename: Jay R
authors.initials: JR
authors.lastname: Winkler
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D003575:Cytochromes c1 / Q000737:chemistry* / Q000378:metabolism*; D004735:Energy Transfer; D019791:Guanidine; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D029701:Saccharomyces cerevisiae Proteins; D003575:Cytochromes c1; D019791:Guanidine