Calcium- and membrane-induced changes in the structure and dynamics of three helical hairpins in annexin B12
Calcium- and membrane-induced changes in the structure and dynamics of three helical hairpins in annexin B12
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DOI：
10.1021/bi051751m
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发表时间：
2005-12-20
期刊：
影响因子：
2.9
通讯作者：
Langen, R
中科院分区：
文献类型：
--
作者：
Isas, JM;Kim, YE;Langen, R
膜联蛋白是一类可溶性蛋白质，可与磷脂膜的界面区域发生可逆的Ca 2+依赖性相互作用。在可溶性膜联蛋白晶体结构的凸面上的螺旋发夹被提出来介导与膜的结合，但其机制尚未确定。在这项研究中，我们使用了一个定点自旋标记（SDSL）的实验方法来研究Ca 2+和膜诱导的结构和动态变化，发生在螺旋发夹涵盖三个四个D和E螺旋的膜联蛋白B12。针对膜联蛋白B 12的以下氮氧化物扫描的可溶性和Ca 2+依赖性膜结合状态分析电子顺磁共振（EPR）参数：第三次重复中D和E螺旋的连续24个残基扫描（残基219-242）的D-E环区域和涵盖第一重复序列（残基68-74）和第四重复序列（300-305）的D-E环区域的短扫描。当蛋白质处于溶液或膜结合状态时，大多数位点的EPR迁移率和可及性参数是相似的，并且两组数据与蛋白质的晶体结构一致。然而，在所有三个环区域中观察到膜诱导的流动性和可及性的变化，在对应于环中高度保守的丝氨酸和甘氨酸残基的位点处注意到最显著的变化。EPR可及性参数清楚地确定，氮氧侧链放置在这些网站直接接触的双层。EPR迁移率参数表明，这些网站是非常移动的在溶液中，但固定在EPR时间尺度上的膜结合状态。由于双层磷脂的头基区域在不存在膜联蛋白的情况下是相对移动的，膜联蛋白B 12的Ca 2+依赖性结合似乎形成复合物，其中蛋白质的D-E环区域和磷脂的头基区域的移动性受到高度限制。膜联蛋白诱导的膜流动性的限制可能的生物学后果进行了讨论。
Annexins are a family of soluble proteins that can undergo reversible Ca2+-dependent interaction with the interfacial region of phospholipid membranes. The helical hairpins on the convex face of the crystal structure of soluble annexins are proposed to mediate binding to membranes, but the mechanism is not defined. For this study, we used a site-directed spin labeling (SDSL) experimental approach to investigate Ca2+ and membrane-induced structural and dynamic changes that occurred in the helical hairpins encompassing three of the four D and E helices of annexin B12. Electron paramagnetic resonance (EPR) parameters were analyzed for the Soluble and Ca2+-dependent membrane-bound states of the following nitroxide scans of annexin B 12: a continuous 24-residue scan of the D and E helices in the third repeat (residues 219-242) and short scans encompassing the D-E loop regions of the first repeat (residues 68-74) and the fourth repeat (300-305). EPR mobility and accessibility parameters of most sites were similar when the protein was in Solution or in the membrane-bound state, and both sets of data were consistent with the crystal structure of the protein. However, membrane-induced changes in mobility and accessibility were observed in all three loop regions, with the most dramatic changes noted at sites corresponding to the highly conserved serine and glycine residues in the loops. EPR accessibility parameters clearly established that nitroxide side chains placed at these sites made direct contact with the bilayer. EPR mobility parameters showed that these sites were very mobile in Solution, but immobilized on the EPR time scale in the membrane-bound state. Since the headgroup regions of bilayer phospholipids are relatively mobile in the absence of annexins, Ca2+-dependent binding of annexin B 12 appears to form a complex in which the mobility of the D-E loop region of the protein and the headgroup region of the phospholipid are highly constrained. Possible biological consequences of annexin-induced restriction of membrane mobility are discussed.