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首页> 外文期刊>Journal of Molecular Biology >From minichaperone to GroEL 3: Properties of an active single-ring mutant of GroEL
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From minichaperone to GroEL 3: Properties of an active single-ring mutant of GroEL

机译：从minichaperone到GroEL 3：GroEL活性单环突变体的性质

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The next step in our reductional analysis of GroEL was to study the activity of an isolated single seven-membered ring of the 14-mer. A known single-ring mutant, GroEL(SR1), contains four point mutations that prevent the formation of double-rings. That heptameric complex is functionally inactive because it is unable to release GroES. We found that the mutation E191G, which is responsible for the temperature sensitive (ts) Escherichia coli allele groEL44 and is located in the hinge region between the intermediate and apical domains of GroEL, appears to function by weakening the binding of GroES, without destabilizing the over all structure of GroEL44 mutant. We introduced, therefore, the mutation E191G into GroEL(SR1) in order to generate a single-ring mutant that may have weaker binding of GroES and hence be active. The new single-ring mutant, GroEL(SR44), was indeed effective in refolding both heat and dithiothreitol-denatured mitochondrial malate dehydrogenase with great efficiency. Further, unlike all smaller constructs of GroEL, the expression of GroEL(SR44) in E. coli that contained no endogenous GroEL restored biological viability, but not as efficiently as does wild-type GroEL. We envisage the notional evolution of the structure and properties of GroEL. The minichaperone core acts as a primitive chaperone by providing a binding surface for denatured states that prevents their self-aggregation. The assembly of seven minichaperones into a ring then enhances substrate binding by introducing avidity. The acquisition of binding sites for Am then allows the modulation of substrate binding by introducing the allosteric mechanism that causes cycling between strong and weak binding sites. This is accompanied by the acquisition by the heptamer of the binding of GroES, which functions as a lid to the central cavity and competes for peptide binding sites. Finally, dimerization of the heptamer enhances its biological activity. (C) 2000 Academic Press. [References: 63]
机译：我们对GroEL进行还原分析的下一步是研究14-mer的一个孤立的单个七元环的活性。已知的单环突变体GroEL（SR1）包含防止双环形成的四个点突变。该七聚体复合物在功能上无活性，因为它无法释放GroES。我们发现，突变E191G负责温度敏感（ts）大肠杆菌等位基因groEL44，位于GroEL中间和顶端结构域之间的铰链区，似乎通过削弱GroES的结合而不破坏其稳定来起作用。 GroEL44突变体的所有结构。因此，我们将突变E191G引入GroEL（SR1），以生成单环突变体，该突变体可能与GroES的结合较弱，因此具有活性。新的单环突变体GroEL（SR44）确实有效地高效折叠了热和二硫苏糖醇变性的线粒体苹果酸脱氢酶。此外，与所有较小的GroEL构建体不同，在不含内源性GroEL的大肠杆菌中GroEL（SR44）的表达可恢复生物活力，但效率不如野生型GroEL。我们设想了GroEL的结构和特性的概念演变。微型分子伴侣核心通过提供变性状态的结合表面来防止其自身聚集，从而充当原始的分子伴侣。然后通过引入亲和力将七个小分子伴侣组装成环。然后，通过引入引起强结合位点和弱结合位点之间循环的变构机制，获得Am结合位点可以调节底物结合。这伴随着七聚体获得GroES的结合，GroES起着盖到中央腔的作用并且竞争肽结合位点。最后，七聚体的二聚化增强了其生物活性。 （C）2000学术出版社。 [参考：63]

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