Furutani M, Iida T, Yoshida T, Maruyama T
Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi-shi, Iwate 026, Japan.
J Biol Chem. 1998 Oct 23;273(43):28399-407. doi: 10.1074/jbc.273.43.28399.
A gene encoding 544 amino acids for a subunit of group II chaperonin (thermosome) was cloned from a thermophilic methanogen, Methanococcus thermolithotrophicus. The deduced amino acid sequence showed 66.5, 56.1, and 20.1% similarities to those of Methanopyrus kandleri and Thermoplasma acidophilum and group I chaperonin of Escherichia coli, respectively. We call this chaperonin MTTS (M. thermolithotrophicus thermosome). The MTTS gene was expressed in E. coli. The purified recombinant MTTS seemed to be monomeric on gel filtration in the absence of Mg2+ and ATP. The monomer assembled to an oligomer (complex) in the presence of 50 mM MgCl2, 0.25 mM ATP, and 0.3 M (NH4)2SO4. It was eluted immediately before the elution volume of E. coli GroEL tetradecamer on gel filtration with a TSKgel G3000SWXL column. This reconstructed MTTS complex showed the cylindrical structure with two stacked rings in electron microscopy. The MTTS complex formed filamentous structures in the presence of Mg2+ and ATP at the protein concentration above 3.0 mg/ml. This filament formation was reversible. The MTTS filament was dissociated to the complex by dilution to the protein concentration of 0.2 mg/ml, even in the presence of Mg2+ and ATP. The MTTS complex exhibited weak ATPase activity with the hydrolysis rate of 74 mol of ATP hydrolysis/mol of MTTS complex/min at 70 degreesC. The MTTS complex promoted the refolding of chemically denatured thermophilic archaeal citrate synthase and glucose dehydrogenase at 50 degreesC in an ATP-dependent fashion. The analysis of nucleotide specificity of chaperone activity of MTTS suggested that it was coupled with hydrolysis of ATP, CTP, or UTP.
从嗜热产甲烷菌嗜热嗜岩石甲烷球菌中克隆出一个编码544个氨基酸的II型伴侣蛋白（热体）亚基的基因。推导的氨基酸序列与坎氏甲烷嗜热菌、嗜酸嗜热放线菌以及大肠杆菌I型伴侣蛋白的氨基酸序列分别具有66.5%、56.1%和20.1%的相似性。我们将这种伴侣蛋白称为MTTS（嗜热嗜岩石甲烷球菌热体）。MTTS基因在大肠杆菌中表达。在没有Mg2+和ATP的情况下，经凝胶过滤纯化的重组MTTS似乎是单体。在50 mM MgCl2、0.25 mM ATP和0.3 M硫酸铵存在的情况下，单体组装成寡聚体（复合物）。在用TSKgel G3000SWXL柱进行凝胶过滤时，它在大肠杆菌GroEL十四聚体的洗脱体积之前立即被洗脱。在电子显微镜下，这种重构的MTTS复合物呈现出具有两个堆叠环的圆柱形结构。在蛋白质浓度高于3.0 mg/ml且存在Mg2+和ATP的情况下，MTTS复合物形成丝状结构。这种丝状结构的形成是可逆的。即使在存在Mg2+和ATP的情况下，将MTTS细丝稀释至蛋白质浓度为0.2 mg/ml时，它也会解离成复合物。MTTS复合物在70℃时表现出较弱的ATP酶活性，水解速率为74摩尔ATP水解/摩尔MTTS复合物/分钟。MTTS复合物在50℃时以ATP依赖的方式促进化学变性的嗜热古菌柠檬酸合酶和葡萄糖脱氢酶的重折叠。对MTTS伴侣活性的核苷酸特异性分析表明，它与ATP、CTP或UTP的水解偶联。
