Folding of apocytochrome c in lipid micelles: formation of alpha-helix precedes membrane insertion.
Apocytochrome c, which in aqueous solution is largely unstructured, acquires a highly alpha-helical structure upon interaction with lipid. The alpha-helix content induced in apocytochrome c depends on the lipid system, and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The folding kinetic mechanism of apocytochrome c induced by zwitterionic micelles of lysophosphatidylcholine (L-PC), predominantly driven by hydrophobic lipid-protein interactions, was investigated by fluorescence stopped-flow measurements of Trp 59 and fluorescein-phosphatidylethanolamine-(FPE) labeled micelles, in combination with stopped-flow far-UV circular dichroism. It was found that formation of the alpha-helical structure of apocytochrome c precedes membrane insertion. The unfolded state in solution (U(W)) binds to the micelle surface in a helical conformation (I(S)) and is followed by insertion into the lipid micelle, i.e., formation of the final helical state H(L). Binding of apocytochrome c to the lipid micelle (U(W) --> I(S)) is concurrent with formation of a large fraction (75-100%, depending on lipid concentration) of the alpha-helical structure of the final lipid-inserted state H(L). The highly helical intermediate I(S) is formed on the time scale of 3-12 ms, depending on lipid concentration, and inserts into the lipid micelle (I(S) --> H(L)) in the time range of approximately 200 ms to >1 s, depending on lipid-to-protein ratio. The final lipid-inserted helical state H(L) in L-PC micelles has an alpha-helix content approximately 65% of that of cytochrome c in solution and has no compact stable tertiary structure as revealed by circular dichroism results.
Bryson
E A
EA
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Rankin
S E
SE
Carey
M
M
Watts
A
A
Pinheiro
T J
TJ
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c; D006736:Horses; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D001059:Apoproteins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D008244:Lysophosphatidylcholines; D008823:Micelles; D014364:Tryptophan; D045304:Cytochromes c