Project Summary Our long-term goal is to understand the mechanism of a class of protein molecules in cell membranes, which enable ions to flow in or out of cells and thereby generate vital cellular electric signals. The knowledge regarding the mechanisms of these proteins, called ion channels, form the essential scientific basis for us to pursue the pathogenic mechanisms of certain disease processes and to develop therapies. Generally, the functions of channels are regulated by specific signaling processes, and what underlie these regulations are protein-conformational changes. The goal of this proposal is to further develop a fluorescence-polarization microscopy method to accurately track in three-dimensions (3D) a fluorophore attached to a protein on an angstrom-and-millisecond scale, and to apply this method to the investigation of conformational dynamics of the bacterial Trk channel, which is a new target to combat certain antibiotics-resistant enterobacteria. Structural biology has yielded abundant protein structures. However, a full understanding of a protein molecule must include both its spatial and temporal features. We thus need to go beyond studying a protein's static structures on an atomic scale and study its dynamics on angstrom-and-millisecond scales. Thus far, the experimental information about protein dynamics is often lacking, due to the absence of relatively general methods for reliably tracking rapid angstrom-scale conformational changes of a protein. Typically, such small changes can be reliably and quantitatively resolved only with structural techniques such as crystallography or Cryo-EM, which, unfortunately, lack time resolution. Light microscopy may be time-resolved but its spatial resolution had remained generally too low to resolve angstrom-scale protein conformational changes. Recently, we have successfully resolved protein conformational changes occurring on millisecond-and- angstrom scales by examining emission polarization of a fluorophore attached to a protein under examination. With a state-of-the-art fluorescence-polarization microscope and an analytic package that we put together, we have achieved an effective angle resolution of 5-10°. Over this range, a rotational motion of a protein molecule of an average size would cause a 1.7 - 3.5 Å change in the chord distance. Here, we will continue to develop this method and demonstrate its applicability beyond the molecule used to develop the method, besides acquiring important knowledge in biomedical science. With this method, we will determine the energetics and kinetics of the protein-conformational changes underling the Trk channel's function. Integrating the resulting dynamic information with available structural information will yield a mechanistic spatiotemporal 4D model that accounts for the channel's behaviors on millisecond-and-angstrom scales. Success of our study will transform the way that we investigate the dynamic mechanisms of proteins, and accelerat...