NMR spectroscopy of α-crystallin.: Insights into the structure, interactions and chaperone action of small heat-shock proteins
NMR spectroscopy of α-crystallin.: Insights into the structure, interactions and chaperone action of small heat-shock proteins
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DOI：
10.1016/s0141-8130(98)00017-8
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发表时间：
1998-05-01
影响因子：
8.2
通讯作者：
Lindner, RA
中科院分区：
文献类型：
--
作者：
Carver, JA;Lindner, RA
与许多小的热休克蛋白（sHsps）一样，α-晶状体蛋白的亚基分子量约为20 kDa，尽管该蛋白质以平均质量约为800 kDa的大聚集体存在。尽管这种大尺寸，观察到α-晶状体蛋白的良好分辨的H-1 NMR光谱，其产生于每个亚基α A-和α B-晶状体蛋白中的短的、极性的、高度柔性的和溶剂暴露的C-末端延伸。在非分子伴侣条件下，这些延伸不参与与其他蛋白质（例如β-和γ-晶体蛋白）的相互作用。如通过NMR研究所确定的突变体的α-A-晶体蛋白与改变其C-末端延伸，延伸有一个重要的作用，作为增溶剂的相对疏水性α-晶体蛋白分子和高分子量（HMW）的复合物，在分子伴侣的行动过程中形成。相关的sHsp，Hsp 25，也表现出灵活的C-末端延伸。在分子伴侣条件下，并且在从旧晶状体分离的HMW复合物中，α A-晶状体蛋白亚基的C-末端延伸保持其灵活性，而α B-晶状体蛋白亚基至少部分地失去其灵活性，这意味着它参与与“底物”蛋白的相互作用。“底物”蛋白质的构象，当它们与α-晶体蛋白相互作用时，已经通过H-1 NMR光谱探测，并且可以得出结论，α-晶体蛋白与处于无序熔融球状态的“底物”蛋白质相互作用，但仅当这种状态是在其大规模聚集和沉淀的途中时。通过对α-晶体蛋白在尿素浓度增加时的H-1和P-31 NMR谱的监测，提出α-晶体蛋白在变性剂存在下呈双结构域结构，较大的C-末端结构域首先解折叠。所有这些数据已被组合成一个模型的四级结构的α-晶体蛋白。该模型有两层，每层大约有40个子单元，排列在一个环形或圆环形。存在一个大的中央空腔，其入口由柔性C端子延伸部环绕。聚集体中的大疏水区域暴露于溶液中，并且可用于在伴侣作用期间与“底物”蛋白质相互作用。(C)1998 Elsevier Science B. V.保留所有权利。
The subunit molecular mass of alpha-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved H-1 NMR spectrum is observed for alpha-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-crystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of alpha A-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic alpha-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'substrate' protein. The conformation of 'substrate' proteins when they interact with alpha-crystallin has been probed by H-1 NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the H-1 and P-31 NMR spectra of alpha-crystallin in the presence of increasing concentations of urea, it is proposed that alpha-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of alpha-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with 'substrate' proteins during the chaperone action. (C) 1998 Elsevier Science B.V. All rights reserved.