Protein folding in classical perspective: folding of horse cytochrome c.

This research article discusses protein folding, specifically focusing on variations of horse ferrocytochrome c. Despite significant differences in stability, these proteins refold at similar rates and to the same form under specific conditions. This study suggests that the folding time for small single-domain proteins is determined by the time the denatured polypeptide needs to find a nativelike topology, rather than the traditional funnel model.

Protein Folding and Stability

• The paper investigates protein folding using two test variants of ferrocytochrome c, a type of protein found in horses. Each of these variants has very different thermodynamic stabilities – the measure of a system’s ability to withstand external disruption.
• Despite the differences in stability, under certain conditions, both variants refold at similar rates and to the same form. The researchers made this observation using neutral (pH 7) and alkaline conditions (pH 12.7), in which the aqueous stabilities were 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), respectively.

Refolding Rates and Conditions

• Through cross-pH studies, where GdnHCl-unfolded states of neutral and alkaline ferrocyt c were allowed to refold, researchers discovered that the refolding rates are largely independent of the stability, configuration, ionization, and solvation of the initial unfolded state.
• This study suggests that the earliest relaxation or reconfiguration of the protein chains is the same and is independent of the initial equilibrium unfolded state, as evidenced by the burst relaxation signals obtained in cross-pH refolding runs showing the same quantitative dependence on GdnHCl.

Transition State and Folding Mechanism

• The study includes an analysis of the transition state of protein folding, suggesting an early transition state where less than a third of the protein surface that is buried in the native state becomes buried again. The energy barrier for this process is roughly around 10 k(B)T.
• The findings suggest that the folding time for small single-domain proteins is determined by the amount of time the denatured polypeptide needs to search for and find a nativelike topology. This conclusion is at odds with the commonly accepted funnel model of protein folding.