New Development of Protein Research Based on Interatomic Cavity

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，通过五个中间体进行动力学循环。DHFR-的绝热可压缩性通过结合或释放这些配体交替改变，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DHFR的三个不同的柔性环中，Gly67、Gly121和Ala145处的单个氨基酸替换导致了压缩性的巨大变化。可压缩性随着酶活性的增加而增加，这表明蛋白质的动力学对功能有积极的贡献。B因子和远离突变部位的空洞受到影响，空洞较多的突变体表现出较大的可压缩性。高压核磁共振显示，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，同时保持整体折叠结构。C-Myb R2亚区的空洞填充突变(V103L)导致压力稳定性显著增加，这为空洞对蛋白质的动力学和稳定性的影响提供了一个明确的例子。通过高压核磁共振首次发现了泛素的两个折叠构象，表明该技术对能量差很小的结构变化非常敏感。这些结果表明，原子间空穴(压力效应)的特征为蛋白质的动力学和功能提供了新的视角。

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.