Getmanova Elena, Patel Ashish B, Klein-Seetharaman Judith, Loewen Michele C, Reeves Philip J, Friedman Noga, Sheves Mordechai, Smith Steven O, Khorana H Gobind
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2004 Feb 3;43(4):1126-33. doi: 10.1021/bi030120u.
Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The (31)P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 degrees C. Below this temperature, the (31)P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 degrees C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, (19)F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the (19)F and the (31)P-labels. The REDOR technique allows (19)F...(31)P distances to be measured out to approximately 12 A with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the (19)F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 A in phosphorylated rhodopsin.
抑制蛋白与光激活的视紫红质的结合涉及对磷酸化C末端以及受体细胞质表面上几个残基的识别。在黑暗、未磷酸化的视紫红质中，这些位点彼此靠近。为了研究磷酸化C末端在活性和非活性受体中的位置和流动性，我们结合了在去污剂胶束中完整哺乳动物光感受器视紫红质的高分辨率溶液和固态核磁共振光谱，并将其作为温度的函数进行研究。视紫红质激酶在C末端尾巴处磷酸化的视紫红质的（31）P核磁共振共振，在使用魔角旋转的单脉冲激发下是可观察到的，直到样品温度达到-40摄氏度。低于这个温度，（31）P共振变宽，只有使用交叉极化才能观察到。这些结果表明，磷酸化的C末端在-40摄氏度以上高度可移动，而在较低温度下则固定不动。为了探测固定化的磷酸化C末端相对于视紫红质细胞质结构域的相对位置，通过视紫红质与2,2,2-三氟乙硫醇（TET）的反应，在位置140和316引入了（19）F标记。固态旋转回波双共振（REDOR）核磁共振用于探测（19）F和（31）P标记之间的核间距离。REDOR技术允许以高分辨率测量（19）F...（31）P距离至约12埃，但在黑暗或光激活后的REDOR实验中未观察到明显的去相。该结果表明，在磷酸化视紫红质中，C末端的磷酸化位点与螺旋8（Cys 316）和第二细胞质环（Cys140）上的（19）F位点之间的距离大于12埃。
