Morris Amie M, Treweek Teresa M, Aquilina J A, Carver John A, Walker Mark J
School of Biological Sciences, University of Wollongong, Australia.
FEBS J. 2008 Dec;275(23):5885-98. doi: 10.1111/j.1742-4658.2008.06719.x.
Small heat shock proteins (sHsps) are intracellular molecular chaperones that prevent the aggregation and precipitation of partially folded and destabilized proteins. sHsps comprise an evolutionarily conserved region of 80-100 amino acids, denoted the alpha-crystallin domain, which is flanked by regions of variable sequence and length: the N-terminal domain and the C-terminal extension. Although the two domains are known to be involved in the organization of the quaternary structure of sHsps and interaction with their target proteins, the role of the C-terminal extension is enigmatic. Despite the lack of sequence similarity, the C-terminal extension of mammalian sHsps is typically a short, polar segment which is unstructured and highly flexible and protrudes from the oligomeric structure. Both the polarity and flexibility of the C-terminal extension are important for the maintenance of sHsp solubility and for complexation with its target protein. In this study, mutants of murine Hsp25 were prepared in which the glutamic acid residues in the C-terminal extension at positions 190, 199 and 204 were each replaced with alanine. The mutants were found to be structurally altered and functionally impaired. Although there were no significant differences in the environment of tryptophan residues in the N-terminal domain or in the overall secondary structure, an increase in exposed hydrophobicity was observed for the mutants compared with wild-type Hsp25. The average molecular masses of the E199A and E204A mutants were comparable with that of the wild-type protein, whereas the E190A mutant was marginally smaller. All mutants displayed markedly reduced thermostability and chaperone activity compared with the wild-type. It is concluded that each of the glutamic acid residues in the C-terminal extension is important for Hsp25 to act as an effective molecular chaperone.
小分子热休克蛋白（sHsps）是细胞内分子伴侣，可防止部分折叠和不稳定蛋白的聚集与沉淀。sHsps包含一个由80 - 100个氨基酸组成的进化保守区域，称为α - 晶状体蛋白结构域，其两侧是序列和长度可变的区域：N端结构域和C端延伸区。尽管已知这两个结构域参与sHsps四级结构的组织及其与靶蛋白的相互作用，但C端延伸区的作用仍不清楚。尽管缺乏序列相似性，但哺乳动物sHsps的C端延伸区通常是一个短的极性片段，无结构且高度灵活，从寡聚结构中突出。C端延伸区的极性和灵活性对于维持sHsps的溶解性及其与靶蛋白的复合都很重要。在本研究中，制备了小鼠Hsp25的突变体，其中C端延伸区第190、199和204位的谷氨酸残基分别被丙氨酸取代。发现这些突变体在结构上发生改变且功能受损。尽管N端结构域中色氨酸残基的环境或整体二级结构没有显著差异，但与野生型Hsp25相比，突变体的暴露疏水性增加。E199A和E204A突变体的平均分子量与野生型蛋白相当，而E190A突变体略小。与野生型相比，所有突变体的热稳定性和伴侣活性均显著降低。得出的结论是，C端延伸区中的每个谷氨酸残基对于Hsp25作为有效的分子伴侣发挥作用都很重要。
