Haley D A, Bova M P, Huang Q L, Mchaourab H S, Stewart P L
Department of Molecular and Medical Pharmacology and Crump Institute for Biological Imaging, UCLA School of Medicine, Los Angeles, CA 90095, USA.
J Mol Biol. 2000 Apr 28;298(2):261-72. doi: 10.1006/jmbi.2000.3657.
The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.
小热休克蛋白（sHSPs）构成了一个多样化的蛋白质家族，在所有生物体中都有产生。它们起着类似伴侣蛋白的作用，即结合未折叠的多肽并防止蛋白质不受控制地聚集。在此，我们展示了对五种不同sHSP组装体的平行冷冻电子显微镜研究：嗜压甲烷球菌HSP16.5、人αB-晶状体蛋白、人HSP27、牛天然α-晶状体蛋白以及αB-晶状体蛋白与未折叠的α-乳白蛋白的复合物。凝胶过滤色谱表明HSP16.5的单分散性最强，而HSP27和α-晶状体蛋白组装体的多分散性更强。颗粒图像显示出类似的趋势，HSP16.5颗粒大多呈现规则且对称的组装体，而HSP27的组装体最不规则，直径范围很广。对颗粒图像进行的对称性测试表明，HSP16.5的八面体对称性比HSP27或α-晶状体蛋白组装体更强。基于5772张施加了八面体对称性的颗粒图像对HSP16.5进行单颗粒重建，得到的结构与晶体结构紧密匹配。此外，冷冻电镜重建揭示了内部密度，推测其对应于晶体结构中未观察到的灵活的32个N端残基。发现N端部分填充了中央腔，这使得HSP16.5不太可能在腔内隔离变性蛋白。与其他接受检查的sHSPs不同，未施加对称性计算得到的重建结果证实了HSP16.5至少存在松散的八面体对称性，而其他sHSPs没有明显的整体对称性。在图像处理过程中增加了质量选择步骤后，对α-晶状体蛋白组装体进行不对称重建，得到的结构分辨率较低。我们将α-晶状体蛋白的重建解释为可变组装体的平均表示，并认为所达到的分辨率表明了可变程度。从冷冻电子显微镜获得的四级结构信息与最近对αA-晶状体蛋白的α-晶状体蛋白结构域折叠和二聚体界面进行的电子顺磁共振研究相关。
