Spooner P J, Watts A
Department of Biochemistry, University of Oxford, U.K.
Biochemistry. 1991 Apr 23;30(16):3880-5. doi: 10.1021/bi00230a011.
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.
进行了³¹P核磁共振测量，以确定牛肉心磷脂与细胞色素c结合时的结构和化学环境。³¹P核磁共振线形推断，大多数脂质保持双层状态，并且脂质磷酸酯的平均构象受与蛋白质结合的影响不大。对水合心磷脂的自旋晶格（T1）弛豫时间随温度的变化分析表明，在约25℃处有一个T1最小值，这导致脂质头部基团中磷酸酯的相关时间为0.71纳秒。蛋白质-脂质复合物的弛豫行为明显不同，磷自旋晶格弛豫率显著增强。蛋白质的这种效应随温度升高而逐渐增加，在高达75℃时未显示T1有最小值。在两种氧化态的蛋白质中均观察到脂质磷T1弛豫的增强，还原态的这种增强不太明显。T1效应的特征以及蛋白质对脂质磷确定的其他弛豫过程（自旋-自旋弛豫和旋转坐标系中的纵向弛豫）的影响表明蛋白质存在强顺磁相互作用。与在“魔角”旋转的样品的弛豫行为比较也与该机制一致。结果表明，细胞色素c与心磷脂双层络合时会发生可逆变性，使得氧化态和还原态蛋白质天然结构中占主导的电子基态可转变为具有更高磁化率的高自旋态。
