Kinetic Studie on the Functional Expression of Chaperonin

伴侣蛋白功能表达的动力学研究

• 批准号: 17370052
• 负责人: KUWAJIMA Kunihiro
• 金额: $ 9.9万
• 依托单位: National Institutes of Natural Sciences Okazaki Research Facilities (2007)The University of Tokyo (2005-2006)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370052/
• 关键词: molecular chaperone; chaperonin; solution X-ray scattering; allosteric transition; folding; ATP binding; 水分子シャペロン; 水分シャペロン; シャペロン

The objective of the present study was to elucidate quantitatively and physicochemically the molecular mechanism of chaperonin functions by means of physical measurements like small-angle X-ray scattering and kinetic measurements like a stopped-flow technique. The following results were obtained.Although the chaperonin GroEL and GroES from Escherichia coli form the GroEL/GroES complex, its stoichiometry, namely, whether they form a 1: 1 molar-ratio bullet-type complex or rather a 1: 2 molar-ratio football-type complex, was not clear. We used the solution X-ray scattering technique to directly observe the structure of the GroEL/GroES complex in solution under a physiological condition. The results have shown that the bullet-type complex is predominant and that the football-type complex is not present in a significant amount.The multi-sigmoidal behavior of the ATP-concentration dependence of the rate constant of the ATP-induced allosteric transition of the chaperonin GroEL has been interpreted in terms of the double-ring structure of GroEL. In the present study, however, we found that a single-ring mutant(SR1) of GroEL exhibited similar multi-sigmoidal behavior of its ATP dependence. This strongly suggests the presence of at least two binding sites for ATP on GroEL. ADP inhibited the ATP-induced allosteric transition of GroEL, and from the dependence of the apparent rate constant of the allosteric transition on ADP, the second ATP-binding site on GroEL was occupied by ADP at 100 μM ADP and 400 μM of ATP. Under the condition but using azido-ADP instead of ADP, we photochemically labeled GroEL. We confirmed the presence of a phosphate group in the photoaffinity labeled GroEL thus obtained using a fluorescent zinc complex, demonstrating the presence of the second ATP-binding site. We are going to identify the location of the second binding site along the amino acid sequence of GroEL.

本研究的目的是通过小角X射线散射等物理测量和停流技术等动力学测量来定量和物理化学地阐明分子伴侣蛋白功能的分子机制。得到以下结果，虽然来自大肠杆菌的伴侣蛋白GroEL和GroES形成GroEL/GroES复合物，但其化学计量，即它们是形成1：1摩尔比的子弹型复合物还是形成1：2摩尔比的足球型复合物，尚不清楚。我们使用溶液X射线散射技术直接观察生理条件下溶液中GroEL/GroES复合物的结构。结果表明，子弹型复合物是占主导地位的，足球型复合物是不存在的显着amount.The多S形行为的ATP浓度依赖的ATP诱导的变构转换的伴侣蛋白GroEL的速率常数已被解释的GroEL的双环结构。然而，在本研究中，我们发现GroEL的单环突变体（SR 1）表现出类似的ATP依赖性的多S形行为。这强烈表明GroEL上存在至少两个ATP结合位点。ADP抑制ATP诱导的GroEL的变构转变，从变构转变的表观速率常数对ADP的依赖性可知，在100 μM ADP和400 μM ATP时，GroEL上的第二个ATP结合位点被ADP占据。在不使用ADP的情况下，我们用叠氮基ADP对GroEL进行了光化学标记。我们证实了磷酸基团的存在下，在光亲和标记的GroEL，从而获得使用荧光锌络合物，证明存在的第二个ATP结合位点。我们将确定第二个结合位点沿着GroEL的氨基酸序列的位置。

项目成果

Folding of Canine Milk Lysozyme: A Ca^<2+>-Binding Lysozyme

犬乳溶菌酶的折叠：Ca^<2>-结合溶菌酶

• 发表时间: 2007
• 作者: KUWAJIMA;Kunihiro
• 通讯作者: Kunihiro

「研究成果報告書概要(和文)」より

摘自《研究结果报告摘要（日文）》

• 发表时间: 2005
• 作者: Kawauchi;et. al.;Nishimura et al.;Dezawa et al.;Yoshizawa et al.;星野 幹雄;星野 幹雄
• 通讯作者: 星野 幹雄

Mutational analysis of protein solubility enhancement using short peptide tags

• DOI: 10.1002/bip.20596
• 发表时间: 2007-01-01
• 期刊: BIOPOLYMERS
• 影响因子: 2.9
• 作者: Kato, Atsushi;Maki, Kosuke;Kuroda, Yutaka
• 通讯作者: Kuroda, Yutaka

Equilibrium and Kinetics ot the Folding and Unfolding of Canine Milk Lysozyme.

犬乳溶菌酶折叠和解折叠的平衡和动力学。

• 发表时间: 2007
• 期刊: Biochemistry 46
• 作者: H. Nakatani;K. Maki;K. Saeki;T. Aizawa;M. Demura;K. Kawano;S. Tomoda &. K. Kuwajima
• 通讯作者: S. Tomoda &. K. Kuwajima

The native and molten globule states of goat α-Iactalbumin characterized by the NMR hydrogen-exchange technique

通过 NMR 氢交换技术表征山羊 α-乳白蛋白的天然状态和熔球状态

• 发表时间: 2007
• 作者: NAKAMURA;Takashi
• 通讯作者: Takashi