RESOLUTION OF INDIVIDUAL LIPIDS IN MIXED PHOSPHOLIPID-MEMBRANES AND SPECIFIC LIPID CYTOCHROME-C INTERACTIONS BY MAGIC-ANGLE-SPINNING SOLID-STATE P-31 NMR
RESOLUTION OF INDIVIDUAL LIPIDS IN MIXED PHOSPHOLIPID-MEMBRANES AND SPECIFIC LIPID CYTOCHROME-C INTERACTIONS BY MAGIC-ANGLE-SPINNING SOLID-STATE P-31 NMR
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DOI：
10.1021/bi00175a014
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发表时间：
1994-03-08
期刊：
影响因子：
2.9
通讯作者：
WATTS, A
中科院分区：
文献类型：
--
作者：
PINHEIRO, TJT;WATTS, A
以二油酰磷脂酰胆碱 (PC)、二油酰磷脂酰乙醇胺 (PE) 和心磷脂 (CL) 以 PC:PE:CL 摩尔比为 2:2:1 构建了线粒体内膜模型，并通过静态和魔角旋转 (MAS) 固态 P-31 NMR 研究了外周膜蛋白细胞色素 c 与该混合膜的相互作用。三组分膜的静态 P-31 NMR 谱是双层结构中磷脂的典型宽粉末模式，是混合膜中每个单独磷脂组分的三个重叠谱的结果，平均有效化学位移各向异性约为 41 ppm。使用魔角旋转 NMR 方法，在狭窄的 MAS P-31 NMR 谱中观察到三个解析共振，每个共振都被分配给混合膜中的每种脂质成分。这使得研究多组分脂质双层中的单个磷脂-蛋白质相互作用成为可能。现在可以评估细胞色素 c 与模型线粒体膜中每种脂质的相互作用。在不存在和存在结合细胞色素 c 的情况下，测量每种磷酸脂的 Phosphorus-31 自旋晶格 (T-1) 弛豫时间作为温度的函数。在分析的温度范围内与细胞色素 c 结合时，T-1 未受到任何脂质的影响。然而，在较低温度下，混合 PC/PE/CL 双层中心磷脂成分的磷 31 化学位移各向异性的平均在细胞色素 c 的结合上不再是轴对称的，而对于 PC 和 PE 成分，轴对称性在本文研究的温度区间内保持不变。结果表明，该模型线粒体膜中细胞色素 c 与心磷脂成分的相互作用最强，但与其他成分的相互作用较小。混合的 CL/PE/PC 双层中的一些脂质侧相分离似乎是由细胞色素 c 的结合诱导的，其中 PE 在磷 31 NMR 时间尺度上分离成各向同性结构。
A model of the inner mitochondrial membrane was constructed with dioleoylphosphatidylcholine (PC), dioleoylphosphatidylethanolamine (PE), and cardiolipin (CL)at a PC:PE:CL molar ratio of 2:2:1, and the interaction of the peripheral membrane protein cytochrome c with this mixed membrane has been investigated by static and magic-angle spinning (MAS) solid-state P-31 NMR. The static P-31 NMR spectrum of the three-component membrane is a typical broad powder pattern for phospholipids in a bilayer structure, and is a result of three overlapping spectra of each individual phospholipid component in the mixed membrane, with an average effective chemical shift anisotropy of approximately 41 ppm. Using magic-angle spinning NMR methods, three resolved resonances are observed in the narrowed MAS P-31 NMR spectrum, each of which has been assigned to each lipid component in the mixed membrane. This allows the investigation of individual phospholipid-protein interactions in multicomponent lipid bilayers. The interaction of cytochrome c with each lipid in a model mitochondrial membrane could now be evaluated. Phosphorus-31 spin-lattice (T-1) relaxation times for each lipid phosphate were measured as a function of temperature, in the absence and presence of bound cytochrome c. T-1 was not affected for any lipid upon binding of cytochrome c over the temperature range analyzed. However, averaging of the phosphorus-31 chemical shift anisotropy for the cardiolipin component in mixed PC/PE/CL bilayers at lower temperatures ceases to be axially symmetric on binding of cytochrome c, while for PC and PE components the axial symmetry is retained over the temperature interval studied here. The results suggest that the strongest interaction of cytochrome c in this model mitochondrial membrane occurs with the cardiolipin component, but less so with the other components. Some lipid lateral-phase separation in the mixed CL/PE/PC bilayer seems to be induced on binding of cytochrome c, in which PE is segregated into isotropic structures on the phosphorus-31 NMR time scale.