title: Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS.
abstract: It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59-heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Sagar V
authors.initials: SV
authors.lastname: Kathuria
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Can
authors.initials: C
authors.lastname: Kayatekin
authors.affiliation: BioCAT, CSRRI, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors.forename: Raul
authors.initials: R
authors.lastname: Barrea
authors.affiliation: BioCAT, CSRRI, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Kondrashkina
authors.affiliation: BioCAT, CSRRI, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors.forename: Rita
authors.initials: R
authors.lastname: Graceffa
authors.affiliation: BioCAT, CSRRI, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Guo
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: R Paul
authors.initials: RP
authors.lastname: Nobrega
authors.affiliation: BioCAT, CSRRI, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Chakravarthy
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
authors.affiliation: BioCAT, CSRRI, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Irving
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: osman.bilsel@umassmed.edu.
authors.forename: Osman
authors.initials: O
authors.lastname: Bilsel
mesh_terms: D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D053838:Scattering, Small Angle
chemical_list: D045304:Cytochromes c
keywords: FRET; protein folding; small-angle X-ray scattering; unfolded state