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New Development of Protein Research Based on Interatomic Cavity

基于原子间腔的蛋白质研究新进展

基本信息

批准号：
12480201
负责人：
GEKKO Kunihiko
金额：
$ 8.96万
依托单位：
Hiroshima University (2001)Kobe University (2000)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate with the aid of coenzyme NADPH in the kinetic reactions cycling through five intermediates. The adiabatic compressibility of DHFR-changes alternatively by binding or releasing these ligands, parallel to the changes in cavity volume, indicating that the cavity plays dominant role in protein dynamics and enzyme function. Single amino acid substitutions at Gly67, Gly121, and Ala145 in three different flexible loops of DHFR induce large changes in compressibility. Compressibility increases with increasing enzyme activity, indicating that protein dynamics positively contributes to the function. The B-factor and cavities at positions far from the mutation sites are influenced and the mutants with a large amount of cavity show large compressibility. High-pressure NMR reveals that pressure induces a local melting of specific structural elements near hydrophobic cavities of the Ras binding domain of RalGDS while the overall folded structure is maintained. A cavity-filling mutation (V103L) of the c-Myb R2 subdomain induces a dramatic increase (in press)ure stability, giving a clear example of the effect of a cavity on dynamics and stability of the protein. Two folded conformers of ubiqutin were first revealed by high pressure-NMR, indicating this technique is very sensitive to the structural change with a small energy difference. These results demonstrate that the characterization of interatomic cavities (pressure effects) gives new insight into protein dynamics and function.

二氢叶酸还原酶（DHFR）在辅酶NADPH的辅助下催化二氢叶酸还原为四氢叶酸，并通过5个中间体循环进行动力学反应。dhfr的绝热压缩性随这些配体的结合或释放交替变化，与空腔体积的变化平行，表明空腔在蛋白质动力学和酶功能中起主导作用。在三种不同的DHFR柔性环中，Gly67、Gly121和Ala145的单氨基酸取代会引起可压缩性的大变化。可压缩性随酶活性的增加而增加，表明蛋白质动力学对其功能有积极作用。远离突变位点的b因子和空腔受到影响，具有大量空腔的突变体表现出较大的压缩性。高压核磁共振表明，压力诱导RalGDS的Ras结合域疏水空腔附近的特定结构元素局部熔化，而整体折叠结构保持不变。c-Myb R2亚结构域的空腔填充突变（V103L）诱导了（在压力下）蛋白质稳定性的急剧增加，这是空腔对蛋白质动力学和稳定性影响的一个清楚的例子。通过高压核磁共振首次发现了两个折叠的泛素构象，表明该技术在能量差很小的情况下对结构变化非常敏感。这些结果表明，原子间空腔（压力效应）的表征为蛋白质动力学和功能提供了新的见解。