Carver J A, Lindner R A, van Boekel M A, Bloemendal H, de Jong W W
Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands.
J Biol Chem. 1996 Nov 15;271(46):29060-6. doi: 10.1074/jbc.271.46.29060.
alpha-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of alpha-crystallin for aggregation-prone proteins is probably based on hydrophobic interactions. alpha-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that alpha-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five alphaA-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of alphaA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.
α-晶体蛋白以多聚体复合物的形式存在，能够抑制未折叠蛋白的沉淀。尽管这种类似伴侣活性的机制尚不清楚，但α-晶体蛋白对易于聚集的蛋白的亲和力可能基于疏水相互作用。α-晶体蛋白向溶液中暴露了相当大的疏水表面，但它们仍然非常稳定且高度可溶。对这一矛盾现象的一种解释可能是，α-晶体蛋白亚基具有极性且无结构的C末端延伸，其起到一种增溶剂的作用。在本文中，我们描述了5种αA-晶体蛋白，其中在C末端延伸中插入了带电荷和疏水的残基。赖氨酸、精氨酸和天冬氨酸的引入对类似伴侣的活性没有实质性影响。相比之下，引入疏水的色氨酸会大大降低功能活性。圆二色性实验表明该突变体具有正常的二级结构，荧光测量显示插入的色氨酸位于极性环境中。然而，核磁共振光谱清楚地表明色氨酸残基的存在显著降低了C末端延伸的灵活性。此外，这种色氨酸的引入导致蛋白质的热稳定性大幅下降。我们得出结论，通过插入高度疏水的残基来改变αA-晶体蛋白C末端延伸的极性会严重干扰结构和功能的完整性。
