title: A two-dimensional view of the folding energy landscape of cytochrome c.
abstract: Time-correlated single photon counting (TCSPC) was combined with fluorescence correlation spectroscopy (FCS) to study the transition between acid-denatured states and the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The use of these techniques in concert proved to be more powerful than either alone, yielding a two-dimensional picture of the folding energy landscape of Cyt c. TCSPC measured the distribution of distances between the heme of the protein and a covalently attached dye molecule at residue C102 (one folding reaction coordinate), whereas FCS measured the hydrodynamic radius (a second folding reaction coordinate) of the protein over a range of pH values. These two independent measurements provide complimentary information regarding protein conformation. We see evidence for a well defined folding intermediate in the acid renaturation folding pathway of this protein reflected in the distribution of lifetimes needed to fit the TCSPC data. Moreover, FCS studies revealed this intermediate state to be in dynamic equilibrium with unfolded structures, with conformational fluctuations into and out of this intermediate state occurring on an approximately 30-micros time scale.
authors.affiliation: Center for Integrated Nanotechnologies and Chemistry Division (C-PCS), Los Alamos National Laboratory, Los Alamos, NM 87545, USA. jwerner@lanl.gov
authors.forename: James H
authors.initials: JH
authors.lastname: Werner
authors.forename: Raymond
authors.initials: R
authors.lastname: Joggerst
authors.forename: R Brian
authors.initials: RB
authors.lastname: Dyer
authors.forename: Peter M
authors.initials: PM
authors.lastname: Goodwin
mesh_terms: D001703:Biophysics / Q000379:methods; D045304:Cytochromes c / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D015233:Models, Statistical; D017785:Photons; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D045304:Cytochromes c