Single-Molecule Fluorescence Studies of a PH Domain: New Insights into the Membrane Docking Reaction
Single-Molecule Fluorescence Studies of a PH Domain: New Insights into the Membrane Docking Reaction
复制标题
DOI：
10.1016/j.bpj.2008.10.020
复制
发表时间：
2009-01-21
影响因子：
3.4
通讯作者：
Falke, Joseph J.
中科院分区：
文献类型：
--
作者：
Knight, Jefferson D.;Falke, Joseph J.
含有膜靶向结构域的蛋白质在许多细胞信号传导途径中发挥着重要作用。然而，膜结合态的重要特征对于本体方法来说是不可见的，从而阻碍了膜靶向反应的机械分析。在这里，我们使用全内反射荧光显微镜 (TIRFM) 结合单粒子追踪，探测从磷酸肌醇通用受体亚型 1 (GRP1) 中分离出的代表性 pleckstrin 同源 (PH) 结构域的膜对接机制。研究结果显示了 GRP1 PH 结构域与含有其罕见目标脂质磷脂酰肌醇 (3,4,5)-三磷酸 [PI(3,4,5)P3] 的膜对接的三个先前未描述的特征。首先，对支持的脂质双层的表面扩散动力学分析表明，在不存在其他阴离子脂质的情况下，PI(3,4,5)P-3 结合蛋白表现出与单个脂质分子相同的扩散常数。其次，阴离子脂质磷脂酰丝氨酸与先前未识别的第二结合位点的结合减慢了扩散和解离动力学。第三，TIRFM 能够直接观察罕见事件，在这些事件中，从膜表面解离后通过溶液进行瞬时扩散，并快速重新结合到附近的膜相关目标脂质。总的来说，这项研究表明体外单分子 TIRFM 为了解膜对接反应的分子机制提供了一个新窗口。
Proteins containing membrane targeting domains play essential roles in many cellular signaling pathways. However, important features of the membrane-bound state are invisible to bulk methods, thereby hindering mechanistic analysis of membrane targeting reactions. Here we use total internal reflection fluorescence microscopy (TIRFM), combined with single particle tracking, to probe the membrane docking mechanism of a representative pleckstrin homology (PH) domain isolated from the general receptor for phosphoinositides, isoform 1 (GRP1). The findings show three previously undescribed features of GRP1 PH domain docking to membranes containing its rare target lipid, phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3]. First, analysis of surface diffusion kinetics on supported lipid bilayers shows that in the absence of other anionic lipids, the PI(3,4,5)P-3-bound protein exhibits the same diffusion constant as a single lipid molecule. Second, the binding of the anionic lipid phosphatidylserine to a previously unidentified secondary binding site slows both diffusion and dissociation kinetics. Third, TIRFM enables direct observation of rare events in which dissociation from the membrane surface is followed by transient diffusion through solution and rapid rebinding to a nearby, membrane-associated target lipid. Overall, this study shows that in vitro single-molecule TIRFM provides a new window into the molecular mechanisms of membrane docking reactions.