Measuring denatured state energetics: Deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

Shubhada Godbole, Barbara Hammack, Bruce E. Bowler

Research output: Contribution to journalArticlepeer-review

Abstract

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heine during early, folding events, consistent with available data in the literature on early folding events for cytochrome c. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)217-228
Number of pages12
JournalJournal of Molecular Biology
Volume296
Issue number1
DOIs
StatePublished - Feb 11 2000

Keywords

  • Cytochrome c
  • Denatured state
  • Guanidine hydrochloride
  • Heme ligation
  • Protein folding

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