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: 6592503
• 负责人: STANLEY J OPELLA
• 金额: $ 17.24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-31 至 2003-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6592503
• 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.

Recoverin是一种23kD的N-肉豆蔻酰化的钙结合蛋白 在脊椎动物的感光细胞中发现。它参与了 视觉兴奋的恢复期和对背景的适应 灯。它通过阻挡光反应来影响光反应 光激发视紫红质的磷酸化。肉豆蔻酰化反应的研究进展 恢复素在钙离子介导的结构中起着关键作用 蛋白质的转换。这种钙-肉豆蔻基开关使 恢复素从胞浆转移到膜上，在那里 在视网膜视杆的调节级联中发挥作用 细胞。报道了未肉豆蔻酰化的恢复素的晶体结构。 单一的钙离子结合可以与溶液的核磁共振结构相比较 根据蛋白质的肉豆蔻酰化的无钙形式测定。两者都有 结构显示出类似的整体折叠，包含4个EF-Hand图案。 这两种形式的螺旋交叉轴存在差异。 蛋白质。这种结构变化可以提供关于 钙-肉豆蔻基开关的分子机制。首字母 钙离子结合的肉豆蔻酰化恢复素的固体核磁共振研究 表格表示在拓扑中存在EF-HAND螺旋 垂直于双层法线。二维PISEMA ~(15)N均一标记肉豆蔻酰化回收率的光谱 二肉豆蔻基磷胆碱(DMPC)双层膜显示大多数 15N化学分子面内螺旋区的共振强度 频移光谱。它证明了这种蛋白质可以机械地 具有肉豆蔻链的玻璃板上的双层取向 将蛋白质锚定在双层中。~(31)P核磁共振波谱证实 双层相对于磁场的取向。 将进行进一步的研究，以计算三维 在脂质双层中插入恢复素的结构以确定 EF-Hand螺旋线的交叉轴。