STRUCTURAL CHARACTERIZATION OF RECOVERIN, CALCIUM BINDING PROTEIN IN VISION

视觉中恢复蛋白、钙结合蛋白的结构表征

• 批准号: 6320894
• 负责人: STANLEY J OPELLA
• 金额: $ 2.06万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6320894
• 关键词: animal tissue; biological products; biomedical resource; eye; nervous system; nuclear magnetic resonance spectroscopy; proteins; structural biology; technology /technique

Recoverin is a 23 kD, N-myristoylated, calcium binding protein found in vertebrate photoreceptor cells. It is involved in the recovery phase of visual excitation and in adaptation to background light. It influences the photoresponse by blocking the phosphorylation of photoexcited rhodopsin. The N-myristoylation of recoverin is found to play a key role in the Ca+ mediated structural switch of the protein. This calcium-myristoyl switch enables the translocation of recoverin from the cytosol to the membrane where it imparts its functioning in the regulatory cascade of the retinal rod cells. The crystal structure of unmyristoylated recoverin, with a single Ca+ bound can be compared to the solution NMR structure determined on the myristoylated Ca+ free form of the protein. Both structures show a similar overall fold containing 4 EF-hand motifs. There is a variation in the helix crossing axis of the two forms of the protein. This structural change can provide information regarding the molecular mechanism of the calcium-myristoyl switch. The initial solid-state NMR studies of myristoylated recoverin in the Ca+ bound form indicate the presence of the EF- hand helices in a topology that lies perpendicular to the bilayer normal. The two-dimensional PISEMA spectrum of uniformly 15N labeled myristoylated recoverin in dimyristoyl phosphocholine (DMPC) bilayers indicates the majority of resonance intensity in the in-plane helical region of the 15N chemical shift spectrum. It demonstrates that the protein can be mechanically oriented in bilayers on glass plates with the myristoyl chain anchoring the protein in the bilayer. 31P NMR spectra confirm the orientation of the bilayer with respect to the magnetic field. Further studies will be conducted to calculate the three-dimensional structure of recoverin inserted in lipid bilayers to determine the crossing axes of the EF-hand helices.

Recovery是一种23 kD的N-肉豆蔻酰化钙结合蛋白 发现于脊椎动物的感光细胞中。 它涉及 视觉兴奋和适应背景的恢复期 光 它通过阻断细胞内的 光激发视紫红质的磷酸化。 的N-豆蔻酰化 recoverin被发现在Ca+介导的结构中起关键作用。 蛋白质的转换。 这种钙-肉豆蔻酰开关使得 恢复素从胞质转移到膜上， 赋予其在视网膜杆的调节级联中的功能 细胞 未豆蔻酰化的恢复素的晶体结构， 单个Ca+结合可以与溶液NMR结构进行比较 基于蛋白质的豆蔻酰化Ca+游离形式确定。 两 结构显示出类似的包含4个EF-手基序的整体折叠。 两种形式的螺旋交叉轴存在变化， 蛋白质。 这种结构变化可以提供有关 钙-肉豆蔻酰转换的分子机制。 初始 肉豆蔻酰化的recoverin在Ca ~（2+）结合态的固体NMR研究 形式表明在拓扑结构中存在EF-手螺旋， 垂直于双层法线。 二维PISEMA 均匀15 N标记肉豆蔻酰化回收蛋白的光谱 二肉豆蔻酰磷酸胆碱（DMPC）双层表明大多数 15 N化学物质的面内螺旋区域中的共振强度 移动光谱 这表明蛋白质可以机械地 在具有肉豆蔻酰链的玻璃板上以双层定向 将蛋白质锚定在双层中。 ~（31）P NMR谱证实了 双层相对于磁场的取向。 将进行进一步的研究，以计算三维 结构的恢复插入脂质双层，以确定 EF-手螺旋的交叉轴。