Probing single-molecule T4 lysozyme conformational dynamics by intramolecular fluorescence energy transfer
Probing single-molecule T4 lysozyme conformational dynamics by intramolecular fluorescence energy transfer
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DOI：
10.1021/jp022406z
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发表时间：
2003-08-07
影响因子：
3.3
通讯作者：
Lu, HP
中科院分区：
文献类型：
--
作者：
Chen, Y;Hu, DH;Lu, HP
我们展示了使用单分子光谱学来研究 T4 溶菌酶在大肠杆菌 B 细胞多糖壁水解反应下的酶构象运动。通过将一对供体-受体染料分子位点特异性地连接到酶上的非干扰位点，通过监测供体-受体发射强度随时间的变化来测量酶的铰链弯曲运动。发现总的酶促反应速率常数在分子与分子之间变化很大。这种静态不均匀性的主要贡献归因于酶在底物上寻找反应位点。我们还应用分子动力学模拟和随机游走模型来分析酶-底物复合物的形成动力学，揭示化学反应过程中的多种中间构象状态。该方法提供了有关微观构象变化漂移速度、扩散系数、摩擦系数、沿反应坐标摩擦消耗的能量以及能量景观的信息。
We demonstrate the use of single-molecule spectroscopy to study enzyme conformational motions of T4 lysozyme under hydrolysis reaction of the polysaccharide walls of E. coli B cells. By attaching a donor-acceptor pair of dye molecules site-specifically to noninterfering sites on the enzyme, the hinge-bending motions of the enzyme are measured by monitoring the donor-acceptor emission intensity as a function of time. The overall enzymatic reaction rate constants are found to vary widely from molecule to molecule. The dominant contribution to this static inhomogeneity is attributed to enzyme searching for reactive sites on the substrate. We have also applied molecular dynamics simulation and a random-walk model to analyze the enzyme-substrate complex formation dynamics, revealing multiple intermediate conformational states in the chemical reaction process. This approach provides information about the microscopic conformational change drifting velocity, diffusion coefficient, friction coefficient, energy consumed by friction along the reaction coordinate, and energy landscape.