Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints
Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints
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DOI：
10.1110/ps.03233703
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发表时间：
2003-11-01
期刊：
影响因子：
8
通讯作者：
Dratz, EA
中科院分区：
文献类型：
--
作者：
Bailey, BW;Mumey, B;Dratz, EA
视紫红质是大G蛋白偶联受体（GPCR）超家族中了解最多的成员。g蛋白扩增级联是由光诱导的视紫红质构象变化触发的，这种变化与其他gpcr中的激动剂引起的变化是同源的。我们利用9种针对视紫红质的单克隆抗体（mab），将“抗体印记”方法应用于天然膜中的光活化视紫红质。这些单抗识别的表位是从噬菌体上显示的随机肽库中选择的。一种新的计算机算法FINDMAP用于将表位映射到紫红质的不连续片段，这些片段在初级序列中距离较远，但在结构上空间接近。FINDMAP发现的n端片段和螺旋VI和螺旋VIII之间的环的邻近与适应黑暗的视紫红质的x射线结构一致。细胞质表面的表位分为两类，具有不同的预测蛋白片段的空间距离，这与抗体在稳定光激视紫红质后视紫红质I或后视紫红质II构象方面的不同偏好相关。稳定后视紫红质II的抗体的表位表明了暗适应视紫红质的构象变化，包括c端尾部的重排和螺旋VI、C-3环的一部分和螺旋VIII的细胞质端暴露的改变。随着更多的抗体受到抗体印迹，这种方法应该提供关于光激发视紫红质构象的越来越详细的信息，并适用于其他具有挑战性的蛋白质靶点的结构研究。
Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.