Insertional mutagenesis as a probe of rhodopsin's topography, stability, and activity.
Insertional mutagenesis as a probe of rhodopsin's topography, stability, and activity.
复制标题
插入诱变作为视紫红质的形貌、稳定性和活性的探针。
DOI：
--
复制
发表时间：
1994
影响因子：
4.8
通讯作者：
J. Nathans
中科院分区：
文献类型：
--
作者：
J. Borjigin;J. Nathans
本文报道了利用灵活表位插入诱变对视紫红质的结构和功能进行的研究。构建了 16 种视紫红质衍生物，每种衍生物均携带源自 c-Myc 蛋白的 12 个氨基酸表位，两侧为五甘氨酸接头。对于八个插入突变体，表位插入物的膜侧面通过完整或透化细胞的免疫染色来确定。结果证实了当前 G 蛋白偶联受体七螺旋模型所假设的六个螺旋连接环以及氨基和羧基末端的侧向性，并为第三个细胞外环的存在提供了第一个实验证据。一般来说，靠近氨基末端或胞外面上的插入物比靠近羧基末端或胞质面上的插入物更有可能破坏折叠和/或稳定性。分别在第二和第三胞质环中的位置139或239处的表位插入未能激活转导蛋白，而在羧基末端尾部的位置333处的插入则具有完全功能。这里描述的实验方法应该证明对于阐明膜蛋白和球状蛋白的结构和功能特性通常有用。
This paper reports a study of rhodopsin's structure and function using insertional mutagenesis with a flexible epitope. Sixteen rhodopsin derivatives were constructed, each of which carried a 12-amino acid epitope derived from the c-Myc protein flanked by penta-glycine linkers. For eight of the insertion mutants, the membrane sideness of the epitope insert was determined by immunostaining of intact or permeabilized cells. The results confirm the sidedness of each of the six helix connecting loops and the amino and carboxyl termini as postulated by the current seven-helix models of G-protein-coupled receptors and provide the first experimental evidence for the existence of the third extracellular loop. In general, inserts that were either closer to the amino terminus or on the extracellular face were more likely to disrupt folding and/or stability than were inserts near the carboxyl terminus or on the cytosolic face. Epitope insertion at positions 139 or 239, in the second and third cytosolic loops, respectively, failed to activate transducin, whereas an insertion at position 333 in the carboxyl-terminal tail was fully functional. The experimental approach described here should prove generally useful for elucidating structural and functional properties of both membrane and globular proteins.