Lu Maolin, Lu H Peter
Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.
J Phys Chem B. 2014 Oct 16;118(41):11943-55. doi: 10.1021/jp5081498. Epub 2014 Oct 3.
Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental understanding of the enzymatic reaction turnover dynamics associated with overall enzyme as well as the specific active-site conformational fluctuations that are not identifiable and resolvable in the conventional ensemble-averaged experiment.
蛋白质的构象运动高度动态且本质上很复杂。为了捕捉构象运动的时间和空间复杂性，并进一步理解它们在蛋白质功能中的作用，人们尝试除了典型的一维荧光共振能量转移（FRET）坐标或在一维FRET坐标上投影的构象运动之外，探测蛋白质的多维构象动力学。通过单分子FRET探测了T4溶菌酶两个结构域之间沿α螺旋的铰链弯曲运动。然而，T4溶菌酶的结构域运动相当复杂，涉及多个耦合的核坐标，并且很可能除了铰链弯曲之外还包含其他运动。超出典型酶促铰链弯曲运动的多维蛋白质构象运动很可能对整体酶促功能有深远影响。在本报告中，我们开发了一种集成了荧光各向异性、FRET和荧光寿命的单分子多参数光子标记光谱学。这种光谱方法能够同时观察单个Cy3 - Cy5标记的T4溶菌酶分子的与FRET相关的位点间构象动力学和分子旋转（或取向）运动。我们通过记录荧光各向异性进一步观察到沿取向坐标广泛分布的旋转灵活性，并通过监测时间依赖性供体寿命同时确定了沿FRET坐标的多个中间构象状态，呈现了在酶促反应条件下T4溶菌酶开闭铰链弯曲酶促周转运动过程中多维构象动力学的全貌。通过分析寿命和各向异性轨迹的自相关函数，我们还观察到了T4溶菌酶多维构象涨落动力学的动态和静态不均匀性，为与整体酶相关的酶促反应周转动力学以及在传统系综平均实验中无法识别和解析的特定活性位点构象涨落提供了基本理解。
