Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Emily J. Guinn, Wayne S. Kontur, Oleg V. Tsodikov, Irina Shkel, M. Thomas Record

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50-90% of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.

Original languageEnglish
Pages (from-to)16784-16789
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number42
StatePublished - Oct 15 2013


  • Collapse
  • Kinetics
  • Native fold
  • Protein denaturation
  • Stability

ASJC Scopus subject areas

  • General


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