Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase.
Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase.
复制标题
识别与磷脂头基接触的膜蛋白区域：一类新的醛脂质标记与细胞色素 c 氧化酶的共价连接。
DOI：
10.1021/bi00349a027
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Griffith,OH
中科院分区：
文献类型：
--
作者：
McMillen,DA;Volwerk,JJ;Ohishi,J;Erion,M;Keana,JF;Jost,PC;Griffith,OH
俄勒冈州大学分子生物学研究所和化学系，尤金，俄勒冈州97403接收1985年6月26日摘要：合成了一系列基于苯甲醛反应基团的胺特异性试剂，并对其进行了表征，并用于研究牛心细胞色素c氧化酶在磷脂双分子层中的重组。该系列包含三类试剂：具有单个烃链的脂溶性磷酸二酯、磷脂类似物和水溶性苯甲醛。所有试剂均经过放射性标记或旋转标记或两者兼而有之。这些苯甲醛与胺形成的席夫碱被认为是可逆的，直到添加还原剂氰基硼氢化钠，而连接的脂质衍生的脂肪醛是不容易可逆的还原剂的情况下。苯甲醛基团提供了一种控制和延迟与完整膜蛋白的永久附着直到重构步骤之后的方便方法。这确保脂质类似物被正确定位以识别脂质-蛋白质界面处的胺基，而不是不加区别地与蛋白质的亲水结构域的胺反应。苯甲醛脂质标记物以高效率附着于细胞色素c氧化酶。通常，存在的脂质标记物的量的20%共价连接至蛋白质，并且每摩尔蛋白质掺入的标记物的摩尔数范围为1至6，这取决于标记物、脂质和蛋白质的摩尔比。由于稀释效应，水溶性苯甲醛的标记效率远低于任何脂质标记所观察到的效率，但通过增加标记浓度实现了等同水平的掺入。含氮氧自由基的磷脂类似物共价连接到重建细胞色素c氧化酶的电子自旋共振光谱表现出一个大的运动限制组件，这是与膜蛋白的疏水表面接触的自旋标记脂质的特征。共价连接的标签和标签自由扩散和接触的蛋白质分子在双层中的线的形状和分裂是相似的，提供了独立的证据，耦合发生在蛋白质-脂质界面。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法研究了苯甲醛试剂在细胞色素c氧化酶多肽组分上的分布。观察到的脂质类似物的标记模式是不受影响的存在下的酰基链上的氮氧部分，但依赖于标记试剂与蛋白质的摩尔比。与脂质标签，带VII是最重的标记，和显着的标记带III，V，和VI观察到在较高的标记率。有很少或没有标记的带I，II和IV。一个不同的标记模式，观察到与水溶性标签，提供额外的证据表明，脂质样苯甲醛试剂与细胞色素c氧化酶从双层的界限。因此，这些新的标记物具有必要的特异性和反应性，可用于将序列数据与膜蛋白的结构和功能相关联，特别是用于鉴定与层状界面处的磷脂头部基团接触的区域。（细胞色素c氧化酶作为细胞呼吸的最后阶段，其中来自氧化食物的电子从细胞色素c转移到分子氧。在这个过程中释放的能量被保存为穿过膜的质子梯度。解决这个复杂结构的问题...
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403 Received June 26, 1985 abstract: A series of amine-specific reagents based on the benzaldehyde reactive group have been syn-thesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible inthe absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin-labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distributionof the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein. With the lipid labels, band VII was the most heavily labeled, and significant labeling of bands III, V, and VI was observed at higher labeling ratios. There was little or no labeling of bands I, II, and IV. A different labeling pattern was observed with the water-soluble label, providing additional evidence that the lipid-like benzaldehyde reagents react with cytochrome c oxidase from the confines of the bilayer. Thus, these new labels have the necessary specificity and reactivity to be useful in correlating sequence data with the structure and function of integral membrane proteins, particularly in identifying regions in contact with phospholipid head groups at the lamellar interface.(Cytochrome c oxidase functions as the final stage of cell respiration where electrons originating from oxidized foodstuffs are transferred from cytochrome c to molecular oxygen. The energy released in this process is conserved as a proton gradient across the membrane. The problem of resolving the structure of this complex …