Competition between reversible aggregation and loop formation in denatured iso-1-cytochrome c

Franco O. Tzul, Eydiejo Kurchan, Heinrich Roder, Bruce E. Bowler

Research output: Contribution to journalArticlepeer-review

Abstract

The competition between intramolecular histidine-heme loop formation and ligand-mediated oligomer formation in the denatured state is investigated for two yeast iso-1-cytochrome c variants, AcH26I52 and AcA25H26I52. Besides the native His 18 heme ligand, both variants contain a single His at position 26. The AcA25H26I52 variant has Pro 25 mutated to Ala. The concentration dependence of the apparent pK a for His 26-heme binding in 3 M guanidine hydrochloride indicates that the P25A mutation disfavors oligomerization mediated by intermolecular heme ligation by 10-fold. Single- and double-pH-jump stopped-flow experiments with the AcH26I52 variant show that fast phases for His-heme bond formation and breakage are due to intramolecular loop formation and slow phases for His-heme bond formation and breakage are due to intermolecular aggregation. The presence of two closely spaced slow phases in the kinetics of loop formation for both variants suggests that intermolecular His 26-heme ligation results in both dimers and higher-order aggregates. The P25A mutation slows formation and speeds breakdown of an initial dimer, demonstrating a strong effect of local sequence on aggregation. Analysis of the kinetic data yields equilibrium constants for intramolecular loop formation and intermolecular dimerization at pH 7.1 and indicates that the rate constant for intermolecular aggregation is very fast at this pH (10 77-10 8 M -1 s -1). In light of the very fast rates of aggregation in the denatured state, comparison of models involving reversible or irreversible oligomerization steps suggests that equilibrium control of the partitioning between folding and aggregation is advantageous for productive protein folding in vivo.

Original languageEnglish
Pages (from-to)481-491
Number of pages11
JournalBiochemistry
Volume48
Issue number2
DOIs
StatePublished - Jan 20 2009

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