Increasing temperature accelerates protein unfolding without changing the pathway of unfolding

Ryan Day, Brian J. Bennion, Sihyun Ham, Valerie Daggett

Research output: Contribution to journalArticlepeer-review

274 Scopus citations


We have traditionally relied on extremely elevated temperatures (498 K, 225°C) to investigate the unfolding process of proteins within the time-scale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298 K to 498 K. The simulation lengths vary from 94 ns to 20 ns, for a total simulation time of 344 ns, or 0.34 μs. At 298 K, the protein is very stable over the full 50 ns simulation. At 348 K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25 ns, explores partially unfolded conformations for 20 ns, and then refolds over the last 35 ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498 K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.

Original languageEnglish (US)
Pages (from-to)189-203
Number of pages15
JournalJournal of Molecular Biology
Issue number1
StatePublished - 2002
Externally publishedYes


  • Activated process
  • Energy landscape
  • Molecular dynamics
  • Protein unfolding
  • Transition state of unfolding

ASJC Scopus subject areas

  • Virology


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