Submillisecond protein folding kinetics studied by ultrarapid mixing

Chi Kin Chan, Yi Hu, Satoshi Takahashi, Denis L. Rousseau, William A. Eaton, James Hofrichter

Research output: Contribution to journalArticle

243 Citations (Scopus)

Abstract

An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent flow created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 μs to 3 ms. To eliminate the heme-ligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The fluorescence decrease is biphasic. There is an unresolved process with τ < 50 μs, followed by a slower, exponential process with τ = 600 μs at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.

Original languageEnglish (US)
Pages (from-to)1779-1784
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number5
DOIs
StatePublished - Mar 4 1997

Fingerprint

Protein Folding
Heme
Fluorescence
Energy Transfer
Guanidine
Cytochromes c
Tryptophan
Ligands
Proteins

Keywords

  • cytochrome c
  • fluorescence
  • Forster energy transfer
  • polymer dynamics

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Submillisecond protein folding kinetics studied by ultrarapid mixing. / Chan, Chi Kin; Hu, Yi; Takahashi, Satoshi; Rousseau, Denis L.; Eaton, William A.; Hofrichter, James.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 5, 04.03.1997, p. 1779-1784.

Research output: Contribution to journalArticle

Chan, Chi Kin ; Hu, Yi ; Takahashi, Satoshi ; Rousseau, Denis L. ; Eaton, William A. ; Hofrichter, James. / Submillisecond protein folding kinetics studied by ultrarapid mixing. In: Proceedings of the National Academy of Sciences of the United States of America. 1997 ; Vol. 94, No. 5. pp. 1779-1784.
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