New Development of Protein Research Based on Interatomic Cavity

二氢叶酸还原酶（DHFR）在辅酶NADPH的帮助下催化二氢叶酸还原为四氢叶酸，在动力学反应中通过五个中间体循环。DHFR-的绝热压缩性通过结合或释放这些配体而交替变化，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Gly 67，Gly 121，和Ala 145在三个不同的柔性环的DHFR单氨基酸取代诱导压缩性的大的变化。可压缩性随着酶活性的增加而增加，表明蛋白质动力学对功能有积极贡献。远离突变位点的B因子和空洞受到影响，空洞数量多的突变体表现出较大的可压缩性。高压NMR表明，压力诱导局部熔化的特定结构元素附近的疏水腔的Ras结合域的RalGDS，而整体折叠结构得以维持。c-Myb R2亚结构域的空腔填充突变（V103 L）诱导了（在压力下）ure稳定性的显著增加，给出了空腔对蛋白质的动力学和稳定性的影响的明确例子。利用高压核磁共振技术首次发现了泛素的两种折叠构象，表明该技术对结构变化非常敏感，能量差很小。这些结果表明，表征原子间的空腔（压力效应）提供了新的见解蛋白质的动力学和功能。

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.