Landin J S, Katragadda M, Albert A D
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Biochemistry. 2001 Sep 18;40(37):11176-83. doi: 10.1021/bi0100539.
The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.
G蛋白偶联受体视紫红质由七个跨膜螺旋组成，这些螺旋埋于脂质双层中，并通过延伸出疏水核心的环结构域相连。使用差示扫描量热法（DSC）研究了视紫红质及其漂白形式视蛋白的热稳定性。热转变是不对称的，且热转变温度取决于扫描速率。这种依赖性表现出两态不可逆变性的特征，其中中间状态迅速转变为最终的不可逆状态。这些研究表明视紫红质和视蛋白的变性受动力学控制。将完整蛋白质的变性与该蛋白质的三种蛋白酶解切割形式进行了比较。胰蛋白酶去除了羧基末端的九个残基，木瓜蛋白酶去除了羧基末端的28个残基以及第三细胞质环的一部分，而胰凝乳蛋白酶切割了细胞质环2和3。在每种情况下，这些片段在膜中仍作为复合物结合在一起。对每种片段化蛋白质进行了DSC研究。在所有样品中，熔解温度（Tm）对扫描速率的依赖性表明该转变受动力学控制。胰蛋白酶处理的蛋白质与完整蛋白质差异不大。然而，当细胞质环3被木瓜蛋白酶或胰凝乳蛋白酶切割时，变性的活化能降低。在漂白和未漂白的样品中均观察到这种情况。在发色团11-顺式视黄醛存在的情况下，木瓜蛋白酶和胰凝乳蛋白酶切割产生的蛋白水解片段之间的非共价相互作用足够强，使得每个复合物都作为一个整体变性。漂白后，木瓜蛋白酶片段表现出单一的热转变。然而，漂白后，胰凝乳蛋白酶片段表现出两个量热转变。这些数据表明视紫红质的环对蛋白质发挥稳定作用。
Zotero 插件