Sakane Isao, Ikeda Mitsuyoshi, Matsumoto Chiduru, Higurashi Takashi, Inoue Katsuaki, Hongo Kunihiro, Mizobata Tomohiro, Kawata Yasushi
Department of Biotechnology, Faculty of Engineering, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Koyama-Minami, Tottori 680-8552, Japan.
J Mol Biol. 2004 Dec 3;344(4):1123-33. doi: 10.1016/j.jmb.2004.09.082.
Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.
伴侣蛋白10（cpn10）是分子伴侣家族中一个高度保守的亚群。GroES是大肠杆菌中的cpn10，由七个10kDa的亚基组成，形成一个圆顶状的寡聚环结构。我们之前的研究发现，GroES通过涉及部分折叠单体的三态解折叠机制完全解折叠，并且该反应是可逆的。为了研究这些解折叠-重折叠特性是否在其他cpn10蛋白中保守，我们检测了大鼠线粒体cpn10（RatES）和嗜热真细菌嗜热栖热菌cpn10（TmaES）的结构稳定性，并将这些值与GroES的值进行比较。在25℃下，在存在各种浓度的盐酸胍的情况下进行尺寸排阻色谱实验，两种cpn10都表现出与GroES相似的解折叠-重折叠特性，即包括形成部分折叠单体的两阶段解折叠反应。尽管与GroES和RatES相比，TmaES的部分折叠单体相当稳定，但发现所有三种cpn10的整体稳定性都通过亚基间相互作用显著实现。我们通过引入一个扰乱亚基缔合的突变，特别是该蛋白N和C末端两条反平行β链之间的相互作用，研究了亚基间相互作用对GroES七聚体整体稳定性的贡献。通过对突变体稳定性的分析，揭示了亚基界面处的反平行β链对于七聚体GroES蛋白的亚基缔合和稳定至关重要。
