REVERSIBLE UNFOLDING OF CYTOCHROME-C UPON INTERACTION WITH CARDIOLIPIN BILAYERS .1. EVIDENCE FROM DEUTERIUM NMR MEASUREMENTS

Deuterium NMR has been used to investigate the structure and dynamic state of cytochrome c complexed with bilayers of cardiolipin. Reductive methylation was employed to prepare [N-epsilon,N-epsilon-C2H3]lysyl cytochrome c, and deuterium exchange provided labeling of backbone sites to give [amide-H-2]cytochrome c or more selective labeling of just histidine residues in [epsilon-H-2]histidine cytochrome c. Deuterium NMR measurements on [N-epsilon,N-epsilon-C2H3]lysyl cytochrome c in the solid state showed restricted motions, fairly typical of the behavior of aliphatic side-chain sites in proteins. The [amide-H-2]cytochrome c provided "immobile" amide spectra showing that only the most stable backbone sites remained labeled in this derivative. Relaxation measurements on the aqueous solution of [amide-H-2]cytochrome c yielded a rotational correlation time of 7.9 ns for the protein, equivalent to a hydrodynamic diameter of 4.0 nm, just 0.6 nm greater than its largest crystallographic dimension. Similar measurements on [epsilon-H-2]histidine cytochrome c in solution showed that all labeled histidine residues were also "immobile" compared with the overall reorientational motion of the protein. The interaction with cardiolipin bilayers appeared to create a high degree of mobility for the side-chain sites of [N-epsilon,N-epsilon-C2H3]lysyl cytochrome c and perturbed backbone structure to instantaneously release all deuterons in [amide-H-2]cytochrome c. The [epsilon-H-2]histidine cytochrome c derivative, when complexed with cardiolipin, failed to produce any detectable wide-line H-2 NMR spectrum, demonstrating that the overall reorientational motion of bound protein was not isotropic on the NMR time scale, i.e., tau-c > 10(-7) s. Additional measurements on the deuterium-exchanged protein-lipid complex, prepared in 2H2O, did not reveal any stable amide sites in the protein backbone, providing the lipid remained in its normal liquid-crystalline state. However, stable backbone sites were detected at reduced temperatures where, according to P-31 NMR observations, the lipid component was becoming immobilized in the complex. A strong binding of protein with liquid-crystalline bilayers of cardiolipin disorders the lysine sites of interaction on the surface of the protein and appears to cause an extensive derangment of secondary structure, such that no stable alpha-helices can exist in the protein backbone with a lifetime longer than around 10(-6) s.