Dynamics and loop function analyses of dihydrofolate reductase

为了阐明蛋白质的结构-动力学-功能关系，我们研究了大肠杆菌二氢叶酸还原酶（DHFR）突变和配体结合对酶功能、绝热压缩率和x射线结构的影响。在三个不同的柔性环中，Gly67、Gly121和Ala145的单氨基酸取代引起了稳定性和绝热压缩性的巨大变化，表明结构的柔韧性得到了改善。远离突变位点的b因子和空腔分布在很大程度上被这些突变改变，表明突变引起的局部结构变化扩展到整个蛋白质分子。突变体的可压缩性随空腔体积的增加而增加，表明原子填充在产生蛋白质动力学中的重要性。虽然突变位点与活性位点很大程度上是分离的，但具有较大可压缩性的突变体表现出较高的酶活性，这主要是由于催化速率的提高。高压核磁共振实验表明，压力对包括辅因子结合域在内的活性位点的柔性环和铰链运动有强烈的影响，高压下两个构象的平衡向开放形式转移。DHFR的可压缩性随着配体结合时腔体体积的变化而变化，反映了反应配位中中间体的柔韧性特征。这些结果表明，由于突变和配体结合引起的局部结构变化通过修饰的原子填充（空腔）影响蛋白质分子的动力学和功能，并且柔性环在蛋白质动力学和功能中起重要作用。

In order to elucidate the structure-dynamics-function relationships of protein, we investigated the effects of mutation and ligand binding of E.Coli dihydrofolate reduactase (DHFR) on the enzymatic function, adiabatic compressibility, and X-ray structure. Single amino acid substitutions at Gly67, Gly121, and Ala145 in three different flexible loops induced large changes in stability and adiabatic compressibility, indicating the modified flexibility of the structure. The B-factor and cavity distribution at sites far from the mutation sites were largely modified by these mutations, indicating that the local changes in structure due to mutation extend to overall of the protein molecule. The compressibility of the mutants increased with increasing the total cavity volume, indicating the importance of atomic packing in generating the protein dynamics. Although the mutation sites are largely separated from the active site, the mutants having a large compressibility showed high enzymatic activity dominantly due to the enhanced catalytic rate. High pressure NMR experiments revealed that the flexible loops and hinge-motion of active-sites including cofactor-binding domain are strongly influenced by pressure and the equilibrium of two conformers is shifted to open form under high pressure. The compressibility of DHFR changed on ligand binding with the change in the cavity volume, reflecting the characteristic flexibility of the intermediates in the reaction coordinate. These results demonstrate that the local structural changes due to mutation and ligand binding affect the dynamics and function of protein molecule through the modified atomic packing (cavity) and that the flexible loops play important role in protein dynamics and function.