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首页> 外文期刊>Biochemistry >Complex of plastocyanin and cytochrome c characterized by NMR chemical shift analysis.
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Complex of plastocyanin and cytochrome c characterized by NMR chemical shift analysis.

机译：质体蓝蛋白和细胞色素c的复合物的特征在于NMR化学位移分析。

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The complexes of horse ferrous and ferric cytochrome c with Cd-substituted pea plastocyanin have been characterized by nuclear magnetic resonance, in order to determine the binding sites and to study the effects of complex formation. Reproducible, small chemical shift changes (0.005-0.05 ppm) were observed for protons in both proteins upon formation of a 1:1 complex. The chemical shift changes depended on the ratio of free to bound protein, with a binding constant of 1.0 +/- 0.5 x 10(5) M(-1), indicating that they were caused by complex formation and that free and bound proteins were in fast exchange. Two-dimensional spectra of the complex of ferrocytochrome c and plastocyanin were screened systematically for chemical shift changes. For about 760 protons, or 70% of the assigned protons in the two proteins, the chemical shift in the complex could be established. In plastocyanin and cytochrome c 14% and 17% of the protons, respectively, showed a significant chemical shift change. These protons form two groups. The first consists of a limited number of surface-exposed side-chain protons. These map on the so-called east side of plastocyanin and the front side of cytochrome c. This group of chemical shift changes is interpreted as representing direct effects of binding, and the respective surfaces thus represent the binding sites. The second group includes backbone amide protons and a few aliphatic and aromatic protons in the hydrophobic core of each protein. The chemical shift changes of this group are interpreted as secondary, i.e., caused by very small structural changes which are transmitted deep into the core of the protein. Ferric cytochrome c caused the same chemical shift effects in plastocyanin as the ferrous form; no intermolecular paramagnetic effects were observed. The small size of the chemical shifts and the absence of intermolecular paramagnetic shifts and NOEs suggest that the complex consists of a dynamic ensemble of structures which are in fast exchange, rather than a single static complex. This study shows that small, reproducible chemical shifts can be used effectively to characterize protein complexes in detail.
机译：为了确定结合位点并研究复合物形成的影响，已通过核磁共振对马来亚铁和三价铁细胞色素c与Cd取代的豌豆质体蓝蛋白的复合物进行了表征。形成1：1的复合物时，两种蛋白质中的质子均具有可重现的小化学位移变化（0.005-0.05 ppm）。化学位移的变化取决于游离蛋白与结合蛋白的比率，结合常数为1.0 +/- 0.5 x 10（5）M（-1），表明它们是由复合物形成引起的，游离蛋白和结合蛋白是快速交换。系统地筛选了铁细胞色素c和质体蓝蛋白复合物的二维光谱，以检测化学位移的变化。对于约760个质子，或两种蛋白质中指定的质子的70％，可以确定复合物中的化学位移。在质体蓝蛋白和细胞色素c中，分别有14％和17％的质子显示出明显的化学位移变化。这些质子形成两组。第一个由有限数量的表面暴露侧链质子组成。这些图位于所谓的质体蓝素的东侧和细胞色素c的前侧。这组化学位移变化被解释为代表结合的直接作用，因此各个表面代表结合位点。第二组包括骨架酰胺质子和每个蛋白质疏水核中的一些脂肪族和芳香族质子。该组的化学位移变化被认为是继发性的，即是由很小的结构变化引起的，该结构变化深入到蛋白质的核心。细胞色素c在质子蓝蛋白中引起的化学位移与亚铁形式相同。没有观察到分子间顺磁效应。化学位移的小尺寸以及分子间顺磁位移和NOE的缺失表明该复合物由快速交换的动态结构组成，而不是单个静态复合物。这项研究表明，小的，可重现的化学位移可以有效地用于详细表征蛋白质复合物。

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