A novel technique will be developed to detect single protein molecules, and to dynamically determine orientations and internal motions of the protein subunits. Single fluorescent molecules bound to the sample proteins with known, pre-determined position and orientation will be observed using the technique of total internal reflection fluorescence microscopy (TIRFM), with highly sensitive, real-time video imaging and solid-state photon counting detectors. This method will be combined, for the first time, with the technique of time-resolved fluorescence polarization (TRFP) to make possible real time quantification of the orientation of single fluorochromes. These modern optical detectors, in conjunction with ultra-clean sample preparations, the background rejection inherent in laser TIRFM and highly efficient collection of sample luminescence will produce a sufficient signal-to-noise ratio of fluorescence emissions to allow reliable recording of angular information from single molecules in the 10 ms time range. The new technology will enable detailed studies of individual biochemical reactions, protein-ligand interactions, and internal motions in single macromolecules. Due to non-uniformities and statistical properties of standard bulk solutions and suspensions, this information can only be obtained from studies of individual molecules. The technology will first be applied to the specific problem of motor proteins that power cell motility. The molecular relationship between biochemical, structural, and mechanical events is a major unresolved question in cell motility. The present proposal impacts directly on this problem by applying a novel optical/physical technique to resolve these events, with an in vitro system that reconstitutes active motility from the purified proteins. The orientation of fluorescent molecules covalently-bound to the light chain region of myosin will be monitored at high time resolution to determine specific structural changes during the functional energy conversion. The methods to be developed will be applicable to virtually every biomolecular system and should have broad impact on biophysical investigations of many enzymes and polynucleotides.
