Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics.
The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.
Pinheiro
T J
TJ
Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK. tpinheiro@bio.warwick.ac.uk
Cheng
H
H
Seeholzer
S H
SH
Roder
H
H
D000577:Amides / Q000378:metabolism; D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D006736:Horses; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity
D000577:Amides; D003574:Cytochrome c Group; D008081:Liposomes; D010743:Phospholipids; D011522:Protons; D012997:Solvents; D006859:Hydrogen