Golden T R, Pedersen P L
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2185, USA.
Biochemistry. 1998 Sep 29;37(39):13871-81. doi: 10.1021/bi981120a.
The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis. To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays. Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity. The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1. A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM. Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein. Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP. Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP. These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site. In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex.
寡霉素敏感性赋予蛋白（OSCP）是线粒体ATP合酶（F0F1）的一个必需亚基，长期以来一直被认为直接参与质子运输与ATP合成的能量偶联。为了深入了解OSCP的功能，在该亚基的一个高度保守的中心区域进行了突变，并使用几种生化分析方法对重组蛋白进行了研究。大鼠肝脏OSCP在大肠杆菌中高水平表达，从包涵体中溶解、复性并纯化至同质。重组蛋白能够将寡霉素敏感的ATP酶活性重构到缺乏F1和OSCP的内膜囊泡中，并以1:1的化学计量比与F1结合。开发了一种新型荧光各向异性分析方法来研究F1与OSCP的结合亲和力，得到的解离常数（Kd）值为51±11 nM。位于OSCP中心区域的两个高度保守的带电荷残基（E91和R94）被突变，重组蛋白（E91Q、R94Q和R94A）被纯化至同质，通过圆二色光谱法判断其结构与野生型蛋白相似。两个R94突变体与F1的结合很少或没有结合，而E91Q的结合方式与野生型OSCP相同。值得注意的是，所有三种突变蛋白都能够与膜重构F1，并赋予与野生型OSCP相同程度的寡霉素敏感性。这些结果表明，分离的大鼠肝脏F1上存在一个与OSCP紧密结合的单一位点，并表明精氨酸94在该位点起关键作用。此外，这些结果表明，将寡霉素敏感性赋予重构的F0F1复合物并不需要这个紧密结合位点。
