Haley D A, Horwitz J, Stewart P L
Department of Molecular & Medical Pharmacology and Crump Institute for Biological Imaging, UCLA School of Medicine, Los Angeles, CA 90095, USA.
J Mol Biol. 1998 Mar 20;277(1):27-35. doi: 10.1006/jmbi.1997.1611.
alphaB-crystallin is a major structural protein in the lens that is found in a variety of other tissues and is associated with numerous neurological disorders. It is a member of the small heat-shock protein family and possesses chaperone-like properties. Cryo-electron microscopy has been applied to analyze the quaternary structure of human recombinant alphaB-crystallin, which spontaneously forms roughly spherical multimers 8 to 18 nm in diameter. Class-sum images based on nearly 5000 alphaB-crystallin particles reveal the presence of a large central cavity, weak regions of density within the protein shell, and an asymmetric quaternary structure. The class-sum images are variable in size and shape, and are suggestive of snapshots of a conformationally flexible assembly. As gel-filtration chromatography reveals a range of molecular masses (650 (+/-140) kDa) for the assembly, the class-sum images were further classified on the basis of total molecular mass. A reconstruction at approximately 4 nm resolution was calculated from the images assigned to 32 subunit (approximately 645 kDa) assemblies. Comparison of class-sum images with reprojections of the reconstruction indicates that the resolution is limited by the variable nature of the assembly. A three-dimensional variance map indicates significant structural divergence within the protein shell and on the outer surface of the particle. Some of the strong variance may correspond to the flexible, exposed C-terminal residues of the alphaB-crystallin monomers. The variable quaternary structure of alphaB-crystallin is consistent with the polydisperse size of the assembly and the previously observed subunit exchange between multimers. Thus, we propose that the monomer packing is variable, and that the quaternary structure of the assembly is not completely defined. A variable alphaB-crystallin quaternary structure may facilitate binding of target proteins in up to stoichiometric ratios.
αB-晶状体蛋白是晶状体中的一种主要结构蛋白，也存在于多种其他组织中，并与多种神经系统疾病相关。它是小分子热休克蛋白家族的成员，具有类似伴侣蛋白的特性。冷冻电子显微镜已被用于分析重组人αB-晶状体蛋白的四级结构，该蛋白能自发形成直径约8至18纳米的大致球形多聚体。基于近5000个αB-晶状体蛋白颗粒的类和图像显示存在一个大的中央腔、蛋白壳内密度较弱的区域以及不对称的四级结构。类和图像在大小和形状上各不相同，提示其为构象灵活的组装体的瞬时图像。由于凝胶过滤色谱显示该组装体的分子量范围为650（±140）kDa，因此根据总分子量对类和图像进行了进一步分类。从分配给32个亚基（约645 kDa）组装体的图像计算出了约4纳米分辨率的重建结构。将类和图像与重建结构的重投影进行比较表明，分辨率受组装体可变性质的限制。三维方差图表明蛋白壳内和颗粒外表面存在显著的结构差异。一些强烈的差异可能对应于αB-晶状体蛋白单体灵活的、暴露的C末端残基。αB-晶状体蛋白可变的四级结构与组装体的多分散大小以及之前观察到多聚体之间的亚基交换一致。因此，我们认为单体堆积是可变的，并且组装体的四级结构并未完全确定。可变的αB-晶状体蛋白四级结构可能有助于以化学计量比结合靶蛋白。
