Senin Ivan I, Churumova Valeriya A, Philippov Pavel P, Koch Karl-Wilhelm
Department of Biology and Environmental Sciences (Biochemistry group), University of Oldenburg, D-26111 Oldenburg, Germany.
BMC Biochem. 2007 Nov 22;8:24. doi: 10.1186/1471-2091-8-24.
The Ca2+-binding protein recoverin operates as a Ca2+-sensor in vertebrate photoreceptor cells. It undergoes a so-called Ca2+-myristoyl switch when cytoplasmic Ca2+-concentrations fluctuate in the cell. Its covalently attached myristoyl-group is exposed at high Ca2+-concentrations and enables recoverin to associate with lipid bilayers and to inhibit its target rhodopsin kinase. At low Ca2+-concentrations the myristoyl group is inserted into a hydrophobic pocket of recoverin thereby relieving inhibitory constraint on rhodopsin kinase. Hydrophobic and electrostatic interactions of recoverin with membranes have not been clearly determined, in particular the function of the positively charged carboxy-terminus in recoverin 191QKVKEKLKEKKL202 in this context is poorly understood.
Binding of myristoylated recoverin to lipid bilayer depends on the charge distribution in phospholipids. Binding was tested by equilibrium centrifugation and surface plasmon resonance (SPR) assays. It is enhanced to a certain degree by the inclusion of phosphatidylserine (up to 60%) in the lipid mixture. However, a recoverin mutant that lacked the charged carboxy-terminus displayed the same relative binding amplitudes as wildtype (WT) recoverin when bound to neutral or acidic lipids. Instead, the charged carboxy-terminus of recoverin has a significant impact on the biphasic dissociation of recoverin from membranes. On the other hand, the nonmyristoylated WT and truncated mutant form of recoverin did not bind to lipid bilayers to a substantial amount as binding amplitudes observed in SPR measurements are similar to bulk refractive index changes.
Our data indicate a small, but evident electrostatic contribution to the overall binding energy of recoverin association with lipid bilayer. Properties of the charged carboxy-terminus are consistent with a role of this region as an internal effector region that prolongs the time recoverin stays on the membrane by influencing its Ca2+-sensitivity.
钙结合蛋白恢复蛋白在脊椎动物光感受器细胞中作为钙传感器发挥作用。当细胞内细胞质钙浓度波动时，它会经历所谓的钙 - 肉豆蔻酰开关。其共价连接的肉豆蔻酰基团在高钙浓度下暴露，使恢复蛋白能够与脂质双层结合并抑制其靶标视紫红质激酶。在低钙浓度下，肉豆蔻酰基团插入恢复蛋白的疏水口袋中，从而解除对视紫红质激酶的抑制性约束。恢复蛋白与膜的疏水和静电相互作用尚未明确确定，特别是在这种情况下，恢复蛋白191QKVKEKLKEKKL202中带正电荷的羧基末端的功能了解甚少。
肉豆蔻酰化的恢复蛋白与脂质双层的结合取决于磷脂中的电荷分布。通过平衡离心和表面等离子体共振（SPR）测定来测试结合。脂质混合物中包含磷脂酰丝氨酸（高达60%）会在一定程度上增强结合。然而，缺乏带电荷羧基末端的恢复蛋白突变体在与中性或酸性脂质结合时，显示出与野生型（WT）恢复蛋白相同的相对结合幅度。相反，恢复蛋白的带电荷羧基末端对恢复蛋白从膜上的双相解离有显著影响。另一方面，非肉豆蔻酰化的WT和截短的恢复蛋白突变体形式与脂质双层的结合量不大，因为SPR测量中观察到的结合幅度与整体折射率变化相似。
我们的数据表明，静电对恢复蛋白与脂质双层结合的总结合能有微小但明显的贡献。带电荷羧基末端的特性与该区域作为内部效应区域的作用一致，该区域通过影响其钙敏感性来延长恢复蛋白在膜上停留的时间。
