A water-lipid interface induces a highly dynamic folded state in apocytochrome c and cytochrome c, which may represent a common folding intermediate.
In this study, we have used CD and NMR techniques to investigate the secondary structure of (apo-) cytochrome c both in solution and when associated with micelles. In aqueous solution, the holoprotein cytochrome c is tightly folded at secondary and tertiary levels and differs strongly from its random-coiled precursor. However, in the presence of 12-PN/12-Pglycol (9:1) micelles, we observed a remarkable resemblance between the CD spectra of these partially helical proteins. The water-lipid interface induces a secondary folding of apocytochrome c, whereas cytochrome c is suggested to partially lose its tertiary structure. The exchange of all amide protons and, using deuterium-labeled proteins, of all amide deuterons with the solvent was monitored by NMR. A rapid exchange rate was observed, indicating that these folding states are highly dynamic. Saturation-transfer NMR of micelle-associated apocytochrome c showed that the exchange takes place at the (sub-) second time scale. The holoprotein in the presence of micelles was found to have two distinct exchange rates: (1) a fast rate, comparable to that found for the micelle-associated precursor and 4.5 times slower than that of the random-coiled apocytochrome c, and (2) a slow rate which is 75 times slower than the precursor in solution. Urea denaturation studies showed the micelle-bound proteins to have a low helix stability, which explains the inability of the lipid-induced secondary structure to prevent its labile protons from rapid exchange. The uniqueness of this lipid-induced highly dynamic folding state of (apo-) cytochrome c is demonstrated by comparison with amphiphilic polypeptides like melittin, and its implications for membrane translocation and functioning are discussed.
de Jongh
H H
HH
Centre for Biomembranes and Lipid Enzymology, University of Utrecht, The Netherlands.
Killian
J A
JA
de Kruijff
B
B
D001059:Apoproteins / Q000737:chemistry*; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D008055:Lipids; D009682:Magnetic Resonance Spectroscopy; D008555:Melitten / Q000737:chemistry; D008823:Micelles; D011487:Protein Conformation; D012996:Solutions; D014867:Water
D001059:Apoproteins; D003574:Cytochrome c Group; D008055:Lipids; D008823:Micelles; D012996:Solutions; D014867:Water; D008555:Melitten; D045304:Cytochromes c; D003903:Deuterium