title: Snapshots of a protein folding intermediate.
abstract: We have investigated the folding dynamics of Thermus thermophilus cytochrome c(552) by time-resolved fluorescence energy transfer between the heme and each of seven site-specific fluorescent probes. We have found both an equilibrium unfolding intermediate and a distinct refolding intermediate from kinetics studies. Depending on the protein region monitored, we observed either two-state or three-state denaturation transitions. The unfolding intermediate associated with three-state folding exhibited native contacts in β-sheet and C-terminal helix regions. We probed the formation of a refolding intermediate by time-resolved fluorescence energy transfer between residue 110 and the heme using a continuous flow mixer. The intermediate ensemble, a heterogeneous mixture of compact and extended polypeptides, forms in a millisecond, substantially slower than the ∼100-μs formation of a burst-phase intermediate in cytochrome c. The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein.
authors: Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.
authors: Seiji
authors: S
authors: Yamada
authors: Nicole D
authors: ND
authors: Bouley Ford
authors: Gretchen E
authors: GE
authors: Keller
authors: William C
authors: WC
authors: Ford
authors: Harry B
authors: HB
authors: Gray
authors: Jay R
authors: JR
authors: Winkler
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D006418:Heme; C022302:cytochrome C-552; D003545:Cysteine