Ghosaini L R, Brown A M, Sturtevant J M
Department of Chemistry, Yale University, New Haven, Connecticut 06511.
Biochemistry. 1988 Jul 12;27(14):5257-61. doi: 10.1021/bi00414a046.
The thermal unfolding of the catabolite activator protein (CAP) of Escherichia coli and the complexes it forms with adenosine cyclic 3',5'-phosphate (cAMP) and guanosine cyclic 3',5'-phosphate (cGMP) was studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of CAP at pH 7.00 gave an irreversible, symmetrical denaturation curve with a single peak. Distinctly different, more complex DSC curves were obtained for the thermal denaturation of the cAMP-protein and cGMP-protein complexes. The DSC data indicate intermolecular cooperation among CAP dimers, with the extent of oligomerization remaining unchanged during unfolding of the protein. The DSC curves for the thermal denaturation of the cAMP-protein complex and cGMP-protein complex have been resolved into three and two components, respectively, according to the model of independent two-state processes. Analysis of the DSC data suggests two and three independent domains for cGMP-protein and cAMP-protein complexes, respectively, with dissociation of mononucleotide occurring in the second component in both cases during protein denaturation. Furthermore, our studies indicate that the presence of either ligand alters the degree of oligomerization of CAP dimers, cAMP having a greater effect than cGMP.
利用高灵敏度差示扫描量热法（DSC）研究了大肠杆菌分解代谢物激活蛋白（CAP）及其与腺苷3',5'-环磷酸（cAMP）和鸟苷3',5'-环磷酸（cGMP）形成的复合物的热解折叠过程。在pH 7.00条件下，CAP的热变性呈现出不可逆的对称变性曲线，且只有一个峰。而cAMP-蛋白复合物和cGMP-蛋白复合物的热变性得到的DSC曲线则明显不同，更为复杂。DSC数据表明CAP二聚体之间存在分子间协同作用，在蛋白质解折叠过程中寡聚化程度保持不变。根据独立双态过程模型，cAMP-蛋白复合物和cGMP-蛋白复合物热变性的DSC曲线分别被解析为三个和两个成分。对DSC数据的分析表明，cGMP-蛋白复合物和cAMP-蛋白复合物分别有两个和三个独立结构域，在蛋白质变性过程中，两种情况下单核苷酸的解离均发生在第二个成分中。此外，我们的研究表明，任一配体的存在都会改变CAP二聚体的寡聚化程度，cAMP的影响比cGMP更大。
