Nuclear magnetic resonance evidence for Ca2+-induced extrusion of the myristoyl group of recoverin
Nuclear magnetic resonance evidence for Ca2+-induced extrusion of the myristoyl group of recoverin
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DOI：
10.1074/jbc.270.52.30909
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发表时间：
1995-12-29
影响因子：
4.8
通讯作者：
Stryer, L
中科院分区：
文献类型：
--
作者：
Ames, JB;Tanaka, T;Stryer, L
Recoverin是新近发现的EF-Hand蛋白超家族成员，在视觉上是一种钙离子感受器，当钙离子水平升高时，与Recoverin氨基末端共价连接的肉豆蔻基或相关的N-酰基使其能够转运到视网膜盘膜上。用二维H-1-C-13移位相关核磁共振谱研究了含有C-13标记的肉豆蔻基的环境，发现在无钙状态下，每对亚甲基质子与肉豆蔻基上的碳原子2、3、11和12键合在一起产生两个峰。由不同亚甲基质子化学位移引起的分裂表明，在无钙状态下，肉豆蔻基与蛋白质发生了密切的相互作用。相反，在钙离子结合的状态下，每对亚甲基质子只有一个峰，这表明肉豆蔻基处于这种形式的各向同性环境中。此外，钙离子结合恢复蛋白的H-1-C-13位移相关谱与肉豆蔻酸在溶液中的H-1-C-13位移相关谱非常相似，我们还利用C-13标记的恢复素进行了H-1-C-13位移相关核磁共振实验，以选择性地探测蛋白质疏水核心中甲基的共振。Ca~(2+)结合的肉豆蔻酰化恢复素与无Ca~(2+)的肉豆蔻酰化恢复素的光谱不同，但与Ca~(2+)结合的非肉豆蔻酸化恢复素的光谱相似。因此，在钙离子结合状态下，肉豆蔻基与肉豆蔻酰化恢复素的疏水核心相互作用很少。用三维(C-13/F-1)编辑(C-13/F-3)-过滤的异核多量子相关-核Overhauser效应光谱，获得了含有C-13标记的肉豆蔻基的恢复素的光谱，以选择性地探测位于肉豆蔻基5埃以内的蛋白质残基。在无钙恢复素中，肉豆蔻基团与许多芳香族残基密切接触，而在钙结合状态下，肉豆蔻基团与蛋白质没有明显的接触。这些核磁共振数据表明，钙离子与恢复素的结合导致其肉豆蔻基被挤出到溶剂中，这将使其能够与目标蛋白的脂双层或疏水部位相互作用。
Recoverin, a recently discovered member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision, A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to translocate to retinal disc membranes when the Ca2+ level is elevated. Two-dimensional H-1-C-13 shift correlation NMR spectra of recoverin containing a C-13-labeled myristoyl group were obtained to selectively probe the effect of Ca2+ on the environment of the attached myristoyl group, In the Ca2+-free state, each pair of methylene protons bonded to carbon atoms 2, 3, 11, and 12 of the myristoyl group gives rise to two peaks. The splittings, caused by nonequivalent methylene proton chemical shifts, indicate that the myristoyl group interacts intimately with the protein in the Ca2+-free state. By contrast, only one peak is seen for each pair of methylene protons in the Ca2+-bound state, indicating that the myristoyl group is located in an isotropic environment in this form. Furthermore, the H-1-C-13 shift correlation NMR spectrum of Ca2+-bound recoverin is very similar to that of myristic acid in solution, H-1-C-13 Shift correlation NMR experiments were also performed with C-13-labeled recoverin to selectively probe the resonances of methyl groups in the hydrophobic core of the protein. The spectrum of Ca2+-bound myristoylated recoverin is different from that of Ca2+-free myristoylated recoverin but similar to that of Ca2+-bound unmyristoylated recoverin. Hence, the myristoyl group interacts little with the hydrophobic core of myristoylated recoverin in the Ca2+-bound state. Three-dimensional (C-13/F-1)-edited (C-13/F-3)-filtered heteronuclear multiple quantum correlation-nuclear Overhauser effect spectroscopy spectra of recoverin containing a C-13-labeled myristoyl group were obtained to selectively probe protein residues located within 5 Angstrom of the myristoyl group. The myristoyl group makes close contact with a number of aromatic residues in Ca2+-free recoverin, whereas the myristoyl group makes no observable contacts with the protein in the Ca2+-bound state. These NMR data demonstrate that the binding of Ca2+ to recoverin induces the extrusion of its myristoyl group into the solvent, which would enable it to interact with a lipid bilayer or a hydrophobic site of a target protein.