Competition between Reversible Aggregation and Loop Formation in Denatured Iso-1-cytochrome c

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 pKa for His 26-heme binding in 3 M gdnHCl 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 (107 to 108 M−1s−1). In light of the very fast rates of aggregation in the denatured state, comparison of models involving reversible or irreversible oligomerization steps suggest that equilibrium control of the partitioning between folding and aggregation is advantageous for productive protein folding in vivo.

Conformational Transitions of Cytochrome c in Sub-Micron-Sized Capsules at Air/Buffer Interface

“…Such bimolecular step is very fast, indicating that intermolecular contacts between denatured proteins occur rapidly even in dilute solution. Such intermolecular contacts have been seen by Tzul et al…”

Formation of Oligomeric Cytochrome c during Folding by Intermolecular Hydrophobic Interaction between N- and C-Terminal α-Helices

“…57 Equilibrium studies of Gdn ion-unfolded yeast iso-1-cyt c showed that intermolecular His26−heme dimers occur in 3 M GdnHCl at pH <5.0 at relatively low protein concentrations. 58 The amount of cyt c oligomers obtained by folding with the desalting method decreased by more than 50% when the pH was decreased from 7.0 to 4.0 (Figure S5 of the Supporting Information), where the amount of bis-His-coordinated species of the unfolded state decreases. 37 These results show that certain amount of domain-swapped dimers may be formed from the bis-His-coordinated species in the unfolded state.…”

“…The unfolded state of cyt c is reported to make a contribution to the stability of the protein . Equilibrium studies of Gdn ion-unfolded yeast iso-1-cyt c showed that intermolecular His26–heme dimers occur in 3 M GdnHCl at pH <5.0 at relatively low protein concentrations . The amount of cyt c oligomers obtained by folding with the desalting method decreased by more than 50% when the pH was decreased from 7.0 to 4.0 (Figure S5 of the Supporting Information), where the amount of bis-His-coordinated species of the unfolded state decreases .…”

Microsecond Folding Dynamics of Apomyoglobin at Acidic pH

“…Progress in protein folding has been closely linked with technical advances in spectroscopy and fast kinetics. T-jump and other laser-initiated techniques − have provided rich kinetic information on the formation of α-helices and β-hairpins, which spans the high nanosecond to low microsecond time range, as well as the folding of some small proteins and domains. − Advances in rapid mixing and detection methods prompted a renaissance of continuous-flow techniques. ,− By coupling an efficient capillary ball mixer with a sensitive charge-coupled device (CCD) camera, we can routinely monitor fluorescence or absorbance changes with a dead time as short as 40 μs, which has yielded a wealth of information on early stages of protein folding. ,− …”