Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, United States.
Biochemistry. 2011 Nov 29;50(47):10399-407. doi: 10.1021/bi201126r. Epub 2011 Nov 2.
The interaction of rhodopsin and transducin has been the focus of study for more than 30 years, but only recently have efforts to purify an activated complex in detergent solution materialized. These efforts have used native rhodopsin isolated from bovine retina and employed either sucrose density gradient centrifugation or size exclusion chromatography to purify the complex. While there is general agreement on most properties of the activated complex, subunit stoichiometry is not yet settled, with rhodopsin/transducin molar ratios of both 2/1 and 1/1 reported. In this report, we introduce methods for preparation of the complex that include use of recombinant rhodopsin, so as to take advantage of mutations that confer constitutive activity and enhanced thermal stability on the protein, and immunoaffinity chromatography for purification of the complex. We show that chromatography on ConA-Sepharose can substitute for the immunoaffinity column and that bicelles can be used instead of detergent solution. We demonstrate the following: that rhodopsin has a covalently bound all-trans-retinal chromophore and therefore corresponds to the active metarhodopin II state; that transducin has an empty nucleotide-binding pocket; that the isolated complex is active and dissociates upon addition of guanine nucleotide; and finally that the stoichiometry corresponds reproducibly to a 1/1 molar ratio of rhodopsin to transducin.
视紫红质与转导蛋白的相互作用已经成为研究焦点 30 多年，但直到最近，人们才努力在去污剂溶液中纯化激活复合物。这些努力使用从牛视网膜中分离的天然视紫红质，并采用蔗糖密度梯度离心或大小排阻色谱法来纯化复合物。尽管人们对激活复合物的大多数性质都达成了共识，但亚基比例尚未确定，报告的视紫红质/转导蛋白摩尔比既有 2/1 也有 1/1。在本报告中，我们介绍了制备复合物的方法，包括使用重组视紫红质，以利用赋予蛋白质组成活性和增强热稳定性的突变，以及免疫亲和色谱法纯化复合物。我们表明，ConA-琼脂糖上的色谱可以替代免疫亲和柱，并且双分子层可以替代去污剂溶液。我们证明了以下几点：视紫红质具有共价结合的全反式视黄醛发色团，因此对应于活性视紫红质 II 状态；转导蛋白具有空的核苷酸结合口袋；分离的复合物是活性的，并在添加鸟嘌呤核苷酸时解离；最后，摩尔比可重复性地对应于视紫红质与转导蛋白的 1/1 摩尔比。
