Structures and Formation Mechanisms of Folding Intermediates of Proteins

蛋白质折叠中间体的结构和形成机制

基本信息

批准号：
06304051
负责人：
KATAOKA Mikio
金额：
$ 6.34万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Co-operative Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06304051/
关键词：
protein folding molten globule X-ray solution scattering protein denturation high pressure NMR stopped-flow CD chaperonine gene engineering 蛋白質変性変性静電的相互作用疎水的相互作用蛋白質非天然構造

项目摘要

Solution structures of molten globules (MG) of various proteins were investigated in detail by solution X-ray scattering. The structure of MG can be classified into two categories : one is close to native structure and the other is composed of a hydrophobic core and flaring tail (s) . Some MG's are stabilized mainly by hydrophobic interaction, the other MG's are stabilized mainly by intramolecular SS bonds. The denatured states cannot be generalized by a term, random coil, because the structural diversity and variety in the denatured states were also indicated.Kinetic studies of beta-lactoglobulin folding demonstrated the accumulation of intermediate with non-native secondary structure, which suggests the importance of non-hierarchical model for folding. Isothermal titration calorimetric measurements on MG formation indicated that the hydrophobic interaction existed in MG is almost 40% of that in native state.It was revealed that denaturant-induced denaturation of alpha-subunit of tryp … More tophane synthase is 3 states, however its thermal denaturation is 2 states. Kinetic studies on proline mutants indicated the existence of two successive intermediates in the folding process of alpha-subunit of tryptophane synthase. Proline residues are contributed to the stability of the late intermediate.The mechanism of target recognition by chaperonine have been investigated using MG of alpha-lactalbumin to reveal the importance of electro-static interaction.Theoretical studies were performed on the global structures of MG and the determinant, especially the contribution of water to the MG formation. Models of MG were proposed to various proteins and the calculated scattering profiles were compared with the observed ones. Consequently the proposed theoretical method was turned out to be quite promising for the folding studies.High pressure NMR method for protein solution was developed and applied to thermal denaturation of RNase A.It was revealed that RNase A undergoes two-state transition even under high pressure. A volume change by denaturation was negative and also a heat capacity at constant pressure was decreased significantly by addition of pressure. Adiabatic compressibility upon denaturation was measured precisely. Less

通过溶液X射线散射详细研究了各种蛋白质的熔球(MG)的溶液结构。MG的结构可分为两类：一类接近天然结构，另一类由疏水核和扩口组成。一些 MG 主要通过疏水相互作用来稳定，其他 MG 主要通过分子内 SS 键来稳定。变性状态不能用术语“无规卷曲”来概括，因为β-乳球蛋白折叠的动力学研究证明了具有非天然二级结构的中间体的积累，这表明非分级模型对于折叠的等温滴定量热测量的重要性。表明 MG 中存在的疏水相互作用几乎是天然状态下的 40%。研究表明，变性剂诱导胰蛋白酶 α 亚基变性……更多托帕烷合酶是3个状态，但其热变性是2个状态。对脯氨酸突变体的动力学研究表明，脯氨酸合酶α亚基折叠过程中两个连续中间体的存在有助于后期中间体的稳定性。利用α-乳清蛋白的 MG 研究了伴侣蛋白的目标识别机制，以揭示静电相互作用的重要性。对 MG 和 MG 的整体结构进行了理论研究。提出了各种蛋白质的决定因素，特别是水对 MG 形成的贡献，并将计算的散射曲线与观察到的散射曲线进行了比较，结果证明所提出的理论方法对于高折叠研究非常有前景。开发了蛋白质溶液的压力核磁共振方法，并将其应用于 RNase A 的热变性。结果表明，即使在高压下，RNase A 也会发生两态转变，变性引起的体积变化为负，并且恒压热容也为负值。精确测量变性时的绝热压缩性。