Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles

Abstract

Binding of native cyt c to L‐PG micelles leads to a partially unfolded conformation of cyt c. This micelle‐bound state has no stable tertiary structure, but remains as α‐helical as native cyt c in solution. In contrast, binding of the acid‐unfolded cyt c to L‐PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L‐PG micelles. Far‐ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle‐associated helical state ( H L ) has a native‐like α‐helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near‐UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid‐unfolded cyt c was investigated by stopped‐flow tryptophan fluorescence. Formation of H L from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle‐induced unfolding of cyt c occurs at a rate ∼0.1 s ∽1 , which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid‐unfolded cyt c with L‐PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.