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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Direct NMR observation of a substrate protein bound to the chaperonin GroEL
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Direct NMR observation of a substrate protein bound to the chaperonin GroEL

机译：与伴侣蛋白GroEL结合的底物蛋白的直接NMR观察

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摘要

The reaction cycle and the major structural states of the molecular chaperone GroEL and its cochaperone, GroES, are well characterized. In contrast, very little is known about the normative states of the substrate polypeptide acted on by the chaperonin machinery. In this study, we investigated the substrate protein human dihydrofolate reductase (hDHFR) while bound to GroEL or to a single-ring analog, SR1, by NMR spectroscopy in solution under conditions where hDHFR was efficiently recovered as a folded, enzymatically active protein from the stable complexes upon addition of ATP and GroES. By using the NMR techniques of transverse relaxation-optimized spectroscopy (TROSY), cross-correlated relaxation-induced polarization transfer (CRIPT), and cross-correlated relaxation-enhanced polarization transfer (CRINEPT), bound hDHFR could be observed directly. Measurements of the buildup of hDHFR NMR signals by different magnetization transfer mechanisms were used to characterize the dynamic properties of the NMR-observable parts of the bound substrate. The NMR data suggest that the bound state includes random coil conformations devoid of stable native-like tertiary contacts and that the bound hDHFR might best be described as a dynamic ensemble of randomly structured conformers.
机译：分子伴侣GroEL及其辅伴分子GroES的反应周期和主要结构状态已得到很好的表征。相反，关于由伴侣蛋白机制作用的底物多肽的规范状态知之甚少。在这项研究中，我们研究了底物蛋白人类二氢叶酸还原酶（hDHFR）在溶液中的NMR光谱结合到GroEL或单环类似物SR1上的条件，其中hDHFR被有效地以折叠的，酶促活性蛋白的形式从HDHFR中回收。加入ATP和GroES后可形成稳定的复合物。通过使用横向弛豫优化光谱（TROSY），互相关弛豫诱导的极化转移（CRIPT）和互相关弛豫增强的极化转移（CRINEPT）的NMR技术，可以直接观察到结合的hDHFR。通过不同的磁化传递机制对hDHFR NMR信号建立的测量用于表征结合底物的NMR可观察部分的动力学特性。 NMR数据表明，结合态包括缺乏稳定的天然样三级接触的无规卷曲构象，结合的hDHFR可能最好描述为随机结构的构象异构体的动态集合。

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