Carver J A, Lindner R A
Department of Chemistry, University of Wollongong, NSW, Australia.
Int J Biol Macromol. 1998 May-Jun;22(3-4):197-209. doi: 10.1016/s0141-8130(98)00017-8.
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 1H 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 1H 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 1H and 31P NMR spectra of alpha-crystallin in the presence of increasing concentrations 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.
α-晶体蛋白的亚基分子量与许多小热休克蛋白（sHsps）一样，约为20 kDa，尽管该蛋白以平均质量约800 kDa的大聚集体形式存在。尽管尺寸很大，但α-晶体蛋白仍能观察到分辨率良好的1H NMR谱，这源于每个亚基（αA-和αB-晶体蛋白）中短的、极性的、高度灵活且暴露于溶剂的C末端延伸。在非伴侣条件下，这些延伸不参与与其他蛋白质（如β-和γ-晶体蛋白）的相互作用。通过对αA-晶体蛋白C末端延伸发生改变的突变体进行NMR研究确定，这些延伸在作为相对疏水的α-晶体蛋白分子和伴侣作用过程中形成的高分子量（HMW）复合物的增溶剂方面具有重要作用。相关的sHsp，Hsp25，也表现出灵活的C末端延伸。在伴侣条件下，以及在从老晶状体中分离出的HMW复合物中，αA-晶体蛋白亚基的C末端延伸保持其灵活性，而αB-晶体蛋白亚基至少部分失去其灵活性，这意味着它参与与“底物”蛋白的相互作用。当“底物”蛋白与α-晶体蛋白相互作用时，其构象已通过1H NMR光谱进行了探测，得出的结论是，α-晶体蛋白与处于无序熔球状态的“底物”蛋白相互作用，但仅当这种状态正朝着大规模聚集和沉淀发展时才会如此。通过监测在尿素浓度增加的情况下α-晶体蛋白的1H和31P NMR谱，有人提出α-晶体蛋白采用两结构域结构，在变性剂存在下，较大的C末端结构域首先展开。所有这些数据已被整合到α-晶体蛋白四级结构的模型中。该模型有两层，每层约40个亚基，排列成环状或环形。存在一个大的中央腔，其入口被灵活的C末端延伸环绕。聚集体中的一个大疏水区域暴露于溶液中，在伴侣作用期间可用于与“底物”蛋白相互作用。
