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E.Coli二氢叶酸还原酶(DHFR)的突变和配体结合对酶功能、绝热压缩性和X射线结构的影响。在三个不同的柔性环上，Gly67、Gly121和Ala145上的单个氨基酸替换导致了稳定性和绝热可压缩性的巨大变化，表明结构的柔性被改变。这些突变在很大程度上改变了远离突变位点的B因子和空洞分布，表明突变引起的局部结构变化延伸到蛋白质分子的整体。突变体的可压缩性随着空腔总体积的增加而增加，表明原子堆积在产生蛋白质动力学中的重要性。虽然突变部位与活性部位很大程度上分离，但压缩性大的突变株表现出较高的酶活性，这主要是由于催化速度的提高。高压核磁共振实验表明，包括辅因子结合区在内的活性中心的柔性环和铰链运动受到压力的强烈影响，两种构象的平衡在高压下向开放状态移动。DHFR的可压缩性随空腔体积的变化而变化，反映了中间体在反应坐标中的柔性特征。这些结果表明，突变和配体结合引起的局部结构变化通过修饰的原子堆积(腔)影响蛋白质分子的动力学和功能，柔性环在蛋白质的动力学和功能中起着重要作用。

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.