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首页> 外文期刊>The journal of physical chemistry, B. Condensed matter, materials, surfaces, interfaces & biophysical >Single-Molecule Nanosecond Anisotropy Dynamics of Tethered Protein Motions
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Single-Molecule Nanosecond Anisotropy Dynamics of Tethered Protein Motions

机译：拴系蛋白运动的单分子纳秒各向异性动力学

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Confined and hindered protein motions generally exist in living cells, with tethered proteins or protein domains particularly associated with and relevant to the early events of molecular interactions in cell signaling at extra- and intracellular membrane surfaces. Ensemble-averaged, time-resolved fluorescence anisotropy has been extensively applied to study the protein rotational and conformational motion dynamics under physiologically relevant conditions. However, the spatial and temporal inhomogeneities of the nonsynchronizable stochastic protein rotational and conformational motions are extremely difficult to characterize with such ensemble-averaged measurements. Here, we demonstrate the use of single-molecule nanosecond anisotropy to study the tethered protein motion of a T4 lysozyme molecule on a biologically compatible surface under water. The rotational motions of tethered proteins are confined in a half-sphere whose volume is primarily defined by the linker and the surface. We observe dynamic inhomogeneities of the rotational diffusion dynamics, i.e., diffusion rate fluctuation, because of interactions between the proteins and the surface. However, we also find that the long-time averages of the dynamically inhomogeneous diffusion rates of single molecules are essentially homogeneous among the single molecules examined. Moreover, tethered proteins stay predominately in solution, rather than being fixed on the modified surface. The infrequent surface interactions are not energetic enough to fix the protein rotational motions. These results suggest that the motions of proteins tethered to surfaces are dynamically inhomogeneous, even if the surfaces or the local environments are homogeneous; in contrast, static inhomogeneity of the rotation dynamics can only exist when the local surface or the local environment are inhomogeneous. Furthermore, the tethered-proteins are found to be in solution without rotational rate fluctuations for most of the time during the measurements, suggesting that the use of tethered proteins on modified glass surfaces under water is a reasonable way to study protein dynamics in solution, as many single-molecule experiments have demonstrated. Our approach allows the recording of time trajectories of the single-molecule rotation rate fluctuation and reveals the single-molecule rotational motion over wide time scale from subnanoseconds to seconds.
机译：受限制和受阻碍的蛋白质运动通常存在于活细胞中，其中拴系的蛋白质或蛋白质结构域与细胞外和细胞内膜表面细胞信号转导中的分子相互作用的早期事件特别相关并相关。集合平均，时间分辨荧光各向异性已被广泛应用于研究生理相关条件下的蛋白质旋转和构象运动动力学。但是，这种同步平均测量很难表征不可同步的随机蛋白质旋转和构象运动在空间和时间上的不均匀性。在这里，我们演示了使用单分子纳秒各向异性来研究水下生物相容性表面上T4溶菌酶分子的束缚蛋白运动。束缚蛋白的旋转运动被限制在一个半球中，其体积主要由连接体和表面决定。由于蛋白质和表面之间的相互作用，我们观察到旋转扩散动力学的动态不均匀性，即扩散速率波动。但是，我们还发现，单个分子的动态不均匀扩散率的长期平均值在所检查的单个分子中基本上是均匀的。而且，拴系蛋白主要留在溶液中，而不是固定在修饰的表面上。罕见的表面相互作用不足以固定蛋白质的旋转运动。这些结果表明，即使表面或局部环境是均质的，与表面相连的蛋白质的运动也是动态不均匀的。相反，旋转动力学的静态不均匀性仅在局部表面或局部环境不均匀时才存在。此外，在测量过程中的大部分时间内，发现束缚蛋白在溶液中的旋转速率没有波动，这表明在水下的改良玻璃表面上使用束缚蛋白是研究溶液中蛋白动力学的合理方法，因为许多单分子实验已经证明。我们的方法允许记录单分子旋转速率波动的时间轨迹，并揭示从纳秒到秒的宽时标上的单分子旋转运动。

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