Swanwick Richard S, Shrimpton Paul J, Allemann Rudolf K
School of Chemistry, University of Birmingham, Edgbaston, UK.
Biochemistry. 2004 Apr 13;43(14):4119-27. doi: 10.1021/bi036164k.
The structure and folding of dihydrofolate reductase (DHFR) from Escherichia coli and the mutant G121V-DHFR, in which glycine 121 in the exterior FG loop was replaced with valine, were studied by molecular dynamics simulations and CD and fluorescence spectroscopy. The importance of residue 121 for the chemical step during DHFR catalysis had been demonstrated previously. High-temperature MD simulations indicated that while DHFR and G121V-DHFR followed similar unfolding pathways, the strong contacts between the M20 loop and the FG loop in DHFR were less stable in the mutant. These contacts have been proposed to be involved in a coupled network of interactions that influence the protein dynamics and promote catalysis [Benkovic, S. J., and Hammes-Schiffer, S. (2003) Science 301, 1196-1202]. CD spectroscopy of DHFR and G121V-DHFR indicated that the two proteins existed in different conformations at room temperature. While the thermally induced unfolding of DHFR was highly cooperative with a midpoint at 51.6 +/- 0.7 degrees C, G121V-DHFR exhibited a gradual decrease in its level of secondary structure without a clear melting temperature. Temperature-induced unfolding and renaturation from the urea-denatured state revealed that both proteins folded via highly fluorescent intermediates. The formation of these intermediates occurred with relaxation times of 149 +/- 4.5 and 256 +/- 13 ms for DHFR and G121V-DHFR, respectively. The fluorescence intensity for the intermediates formed during refolding of G121V-DHFR was approximately twice that of the wild-type. While the fluorescence intensity then slowly decayed for DHFR toward a state representing the native protein, G121V-DHFR appeared to be trapped in a highly fluorescent state. These results suggest that the reduced catalytic activity of G121V-DHFR is the consequence of nonlocal structural effects that may result in a perturbation of the network of promoting motions.
通过分子动力学模拟以及圆二色光谱（CD）和荧光光谱研究了来自大肠杆菌的二氢叶酸还原酶（DHFR）及其突变体G121V - DHFR的结构和折叠情况。在该突变体中，位于外部FG环的甘氨酸121被缬氨酸取代。先前已证明残基121在DHFR催化过程中的化学步骤中具有重要性。高温分子动力学模拟表明，虽然DHFR和G121V - DHFR遵循相似的去折叠途径，但在突变体中，DHFR中M20环与FG环之间的强相互作用不太稳定。有人提出这些相互作用参与了一个影响蛋白质动力学并促进催化作用的耦合相互作用网络[本科维奇，S. J.，和哈姆斯 - 希弗，S.（2003年）《科学》301，1196 - 1202]。DHFR和G121V - DHFR的圆二色光谱表明，这两种蛋白质在室温下以不同构象存在。虽然DHFR的热诱导去折叠具有高度协同性，中点温度为51.6±0.7℃，但G121V - DHFR的二级结构水平逐渐降低，没有明确的解链温度。温度诱导的去折叠以及从尿素变性状态的复性表明，这两种蛋白质都通过高度荧光的中间体进行折叠。对于DHFR和G121V - DHFR，这些中间体的形成分别具有149±4.5和256±13毫秒的弛豫时间。G121V - DHFR复性过程中形成的中间体的荧光强度约为野生型的两倍。虽然DHFR的荧光强度随后缓慢衰减至代表天然蛋白质的状态，但G121V - DHFR似乎被困在高度荧光的状态。这些结果表明，G121V - DHFR催化活性降低是可能导致促进运动网络受到扰动的非局部结构效应的结果。
