Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

Abstract

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.

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

“…1F, Upper). Simultaneous measurements of photon chronic arrival time and delay time along with rotational anisotropy improve the resolution of detection of individual species and provide rich information about the properties of a fluorophore and its surrounding environment (9,(31)(32)(33)(34)(35)(36). In this regard our study is unique and advanced in interrogating the productreleasing mechanism and associated conformational dynamics.…”

“…To investigate in detail, we have plotted the 2D joint distribution of lifetime (τ f ) and anisotropy (r) (32,41). Fig.…”

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease

“…2. It is reasonable to interpret the number of peaks in the probability distribution profile as the number of discrete conformations sampled by the fluorophore (55,56) and to assume that the peak areas reflect their relative populations. The TF decay profile of unliganded sTrp-GCK was best fit by a four-exponential decay plus baseline (Fig.…”

Out-of-Equilibrium Biophysical Chemistry: The Case for Multidimensional, Integrated Single-Molecule Approaches

“… 75 − 77 Therefore, reducing a multidimensional energy landscape into 1-D does not adequately represent the system’s dynamics and can mask physiologically relevant accessible conformations, configurations, and transition paths. 78 , 79 …”