Single-molecule nanosecond anisotropy dynamics of tethered protein motions

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.