Order of Steps in the Cytochrome c Folding Pathway: Evidence for a Sequential Stabilization Mechanism

Mallela M G Krishna, Haripada Maity, Jon N. Rumbley, Yan Lin, S. Walter Englander

Research output: Contribution to journalArticlepeer-review

88 Scopus citations


Previous work used hydrogen exchange (HX) experiments in kinetic and equilibrium modes to study the reversible unfolding and refolding of cytochrome c (Cyt c) under native conditions. Accumulated results now show that Cyt c is composed of five individually cooperative folding units, called foldons, which unfold and refold as concerted units in a stepwise pathway sequence. The first three steps of the folding pathway are linear and sequential. The ordering of the last two steps has been unclear because the fast HX of the amino acid residues in these foldons has made measurement difficult. New HX experiments done under slower exchange conditions show that the final two foldons do not unfold and refold in an obligatory sequence. They unfold separately and neither unfolding obligately contains the other, as indicated by their similar unfolding surface exposure and the specific effects of destabilizing and stabilizing mutations, pH change, and oxidation state. These results taken together support a sequential stabilization mechanism in which folding occurs in the native context with prior native-like structure serving to template the stepwise formation of subsequent native-like foldon units. Where the native structure of Cyt c requires sequential folding, in the first three steps, this is found. Where structural determination is ambiguous, in the final two steps, alternative parallel folding is found.

Original languageEnglish (US)
Pages (from-to)1410-1419
Number of pages10
JournalJournal of Molecular Biology
Issue number5
StatePublished - Jun 23 2006

Bibliographical note

Funding Information:
This work was supported by NIH research grant GM031847.


  • folding pathway
  • hydrogen exchange
  • m value
  • mutation
  • stability labeling


Dive into the research topics of 'Order of Steps in the Cytochrome c Folding Pathway: Evidence for a Sequential Stabilization Mechanism'. Together they form a unique fingerprint.

Cite this