STRUCTURAL AND KINETIC DESCRIPTION OF CYTOCHROME-C UNFOLDING INDUCED BY THE INTERACTION WITH LIPID VESICLES

The interaction of cytochrome c with anionic lipid vesicles of DOPS in duces an extensive disruption of the native structure of the protein. The kinetics of this lipid-induced unfolding process were investigated in a series of fluorescence-and absorbance-detected stopped-flow meas urements. The results show that the tightly packed native structure of cytochrome c is disrupted at a rate of similar to 1.5 s(-1) (independ ent of protein and lipid concentration), leading to the formation of a lipid-inserted denatured state (D-L). Comparison with the expected ra te of unfolding in solution (similar to 2 x 10(-3) s(-1) at pH 5.0 in the absence of denaturant) suggests that the lipid environment dramati cally accelerates the structural unfolding process of cytochrome c. We propose that this acceleration is in part due to the low effective pH in the vicinity of the lipid headgroups. This hypothesis was tested b y comparative kinetic measurements of acid unfolding of cytochrome c i n solution. Our absorbance and fluorescence kinetic data, combined wit h a well-characterized mechanism for folding/unfolding of cytochrome c in solution, allow us to propose a kinetic mechanism for cytochrome c unfolding at the membrane surface. Binding of native cytochrome c in water (N-W) to DOPS vesicles is driven by the electrostatic interactio n between positively charged residues in the protein and the negativel y charged lipid headgroups on the membrane surface. This binding step occurs within the dead time of the stopped-flow experiments (<2 ms), w here a membrane-associated native state (NS) is formed. Unfolding of N S driven by the acidic environment at the membrane surface is proposed to occur via a native-like intermediate lacking Met 80 ligation (M-S) , as previously observed during unfolding in solution. The overall unf olding process (N-S-->D-L) is limited by the rate of disruption of the hydrophobic core in M-S. Equilibrium spectroscopic measurements by ne ar-IR and Soret absorbance, fluorescence, and circular dichroism showe d that D-L has native-like helical secondary structure, but shows no e vidence for specific tertiary interactions. This lipid-denatured equil ibrium state (D-L) is clearly more extensively unfolded than the A-sta te in solution, but is distinct from the unfolded protein in water (U- W), which has no stable secondary structure.