Paci E, Caflisch A, Plückthun A, Karplus M
Laboratoire de Chimie Biophysique Institut Le Bel, Université Louis Pasteur, 4 rue Blaise Pascal, Strasbourg, 67000, France.
J Mol Biol. 2001 Nov 30;314(3):589-605. doi: 10.1006/jmbi.2001.5103.
The unbinding of fluorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigated by biased molecular dynamics with an implicit solvation model. To obtain statistically meaningful results, a large number of unbinding trajectories are calculated; they involve a total simulation time of more than 200 ns. Simulations are carried out with a time-dependent perturbation and in the presence of a constant force. The two techniques, which provide complementary information, induce unbinding by favoring an increase in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates unfolding pathways that are close to equilibrium and can be used to reconstruct the mean force; i.e. the derivative of the potential of mean force, along the reaction coordinate. This is supported by an analysis of the overall unbinding profile and the magnitude of the mean force, which are similar to those of the unbinding force (i.e. the external force due to the time-dependent perturbation) averaged over several unbinding events. The multiple simulations show that unbinding proceeds along a rather well-defined pathway for a broad range of effective pulling speeds. Initially, there is a distortion of the protein localized in the C-terminal region followed by the fluorescein exit from the binding site. This occurs in steps that involve breaking of specific electrostatic and van der Waals interactions. It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for different regimes. Based on the unbinding events, mutations in the 4D5Flu antigen binding site are predicted to result in significant changes in the unbinding force.
利用隐式溶剂化模型通过有偏分子动力学研究了荧光素从4D5Flu抗体单链Fv片段上的解离。为了获得具有统计意义的结果，计算了大量的解离轨迹；它们的总模拟时间超过200纳秒。模拟是在随时间变化的微扰和恒定力的作用下进行的。这两种技术提供了互补信息，通过促进配体与抗体之间距离的增加来诱导解离。该距离是解离反应的一个合适的进展变量，并且允许将模拟中的解离力与原子力显微镜（AFM）数据进行直接比较。随时间变化的微扰产生接近平衡的展开路径，可用于重建平均力；即沿反应坐标的平均力势的导数。对整体解离轮廓和平均力大小的分析支持了这一点，它们与在几个解离事件上平均的解离力（即由于随时间变化的微扰产生的外力）的轮廓和大小相似。多次模拟表明，对于广泛的有效拉动速度，解离沿着相当明确的路径进行。最初，蛋白质在C端区域发生扭曲，随后荧光素从结合位点逸出。这是通过涉及特定静电和范德华相互作用断裂的步骤发生的。由于前者的展开速度要高得多，模拟似乎没有探索与AFM实验中测量的相同的障碍。力对加载速率对数的依赖性是线性的，并且斜率高于AFM中的斜率，这与其他系统中的实验一致，在其他系统中不同区域观察到了不同的斜率。基于解离事件，预测4D5Flu抗原结合位点的突变将导致解离力发生显著变化。
