GSBS Student Publications
Title
Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS
GSBS Program
Biochemistry & Molecular Pharmacology
Publication Date
2014-05-01
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology
Document Type
Article
Disciplines
Molecular Biology
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 Forster 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 mus of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Forster 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.
DOI of Published Version
10.1016/j.jmb.2014.02.020
Source
J Mol Biol. 2014 May 1;426(9):1980-94. doi: 10.1016/j.jmb.2014.02.020. Epub 2014 Mar 4. Link to article on publisher's site
Journal/Book/Conference Title
Journal of molecular biology
Related Resources
PubMed ID
24607691
Repository Citation
Kathuria SV, Kayatekin C, Barrea RA, Kondrashkina E, Graceffa R, Guo L, Nobrega RP, Chakravarthy S, Matthews CR, Irving TC, Bilsel O. (2014). Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS. GSBS Student Publications. https://doi.org/10.1016/j.jmb.2014.02.020. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/2051