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

Area of Science:

• Biochemistry
• Protein Folding
• Molecular Biology

Background:

• Investigating the denatured state of proteins is crucial for understanding folding pathways.
• Yeast iso-1-cytochrome c variants were used to study intramolecular loop formation versus intermolecular oligomerization.
• The role of histidine-heme ligation in protein aggregation was examined.

Purpose of the Study:

• To investigate the competition between intramolecular loop formation and intermolecular oligomerization in denatured yeast iso-1-cytochrome c.
• To determine the effect of a Proline 25 to Alanine (P25A) mutation on these processes.
• To analyze the kinetics and equilibrium of histidine-heme ligation and aggregation.

Main Methods:

• Utilized two yeast iso-1-cytochrome c variants: AcH26I52 and AcA25H26I52.
• Employed concentration-dependent pK(a) measurements in guanidine hydrochloride.
• Performed single- and double-pH-jump stopped-flow experiments.

Main Results:

• The P25A mutation disfavored intermolecular heme ligation-mediated oligomerization by 10-fold.
• Fast kinetics in stopped-flow experiments corresponded to intramolecular loop formation, while slow phases indicated intermolecular aggregation (dimers and higher-order aggregates).
• The P25A mutation slowed dimer formation and accelerated its breakdown, highlighting local sequence effects on aggregation.

Conclusions:

• Intramolecular loop formation and intermolecular aggregation are in competition in the denatured state.
• Local sequence, specifically the P25A mutation, significantly impacts aggregation kinetics.
• Equilibrium control of partitioning between folding and aggregation is advantageous for productive protein folding in vivo, given the rapid aggregation rates.