Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 2. Evidence from phosphorus-31 NMR measurements.
31P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. 31P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T1) relaxation times of hydrated cardiolipin as a function of temperature describes a T1 minimum at around 25 degrees C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behavior of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T1 up to 75 degrees C. The enhancement in lipid phosphorus T1 relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T1 effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the "magic angle" was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.
Spooner
P J
PJ
Department of Biochemistry, University of Oxford, U.K.
Watts
A
A
D002308:Cardiolipins / Q000737:chemistry / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010758:Phosphorus; D011487:Protein Conformation; D013816:Thermodynamics
D002308:Cardiolipins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D010758:Phosphorus