Conformational properties of the iso-1-cytochrome c denatured state: Dependence on guanidine hydrochloride concentration

Eydiejo Wandschneider, Bruce E. Bowler

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27 Scopus citations


Production of seven single surface histidine variants of yeast iso-1-cytochrome c allowed measurement of the apparent pKa, pK a(obs), for histidine-heme loop formation for loops of nine to 83 amino acid residues under varying denaturing conditions (2 M to 6 M guanidine hydrochloride, gdnHCl). A linear correlation between pKa(obs) and the log of the loop size is expected for a random coil, pKa(obs) ∝klog(n), where k is a scaling factor and n is the number of monomers in the loop. For small loops of nine, 16, and 22 monomers, no dependence of pK a(obs) on loop size was observed at any denaturant concentration indicating effects from chain stiffness. For larger loops of 37, 56, 72, and 83 monomers, the dependence of pKa(obs) on log(n) was linear and the slope of that dependence decreased with increasing concentration of denaturant. The scaling factor obtained at 5 M and 6 M gdnHCl for the larger loop sizes was ∼-2.0, close to the value of -2.2 expected for a random coil with excluded volume. However, scaling factors obtained under less harsh denaturing conditions (2 M to 4.5 M gdnHCl) deviated strongly from that expected for a random coil, being in the range -3 to -4. The gdnHCl dependence of pKa(obs) at each loop size was also evaluated to obtain denaturant m-values. Short loops where chain stiffness dominates had similar m-values of ∼0.25kcal/molM. For larger loops m-values decrease with increasing loop size indicating that less hydrophobic area is sequestered when larger loops form. It is known that the earliest events in protein folding involve the formation of simple loops. The data from these studies provide direct insight into the relative probability with which loops of different sizes will form, as well as the factors which affect loop formation.

Original languageEnglish
Pages (from-to)185-197
Number of pages13
JournalJournal of Molecular Biology
Issue number1
StatePublished - May 21 2004


  • cytochrome c
  • denatured states
  • guanidine hydrochloride
  • protein folding
  • random coil


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