Physics Colloquium
Monday, February 28th, 2005, 4:00 P.M.

Ivan Rasnik

University of Illinois at Urbana-Champaign

Understanding the mechanisms of molecular motors: Single molecule Fluorescence Resonant Energy Transfer experiments

Single molecule Fluorescence Resonant Energy Transfer (smFRET) has proved to be a powerful technique to study biological systems. The possibility of look at single molecules eliminates the limitations inherent to bulk experiments while the use of FRET as a nanoscopic ruler allows for a spatial resolution that can not be achieved with other techniques. Helicases are enzymes that catalyze the unwinding of double stranded DNA into single stranded DNA. These proteins are able to couple chemical energy obtained from tri-phosphate nucleotides (as ATP) into mechanical work necessary for unidirectional translocation on the DNA substrate and strand separation. The understanding at a molecular level of translocase and helicase activity requires the knowledge of the kinetic steps involved in the reaction and the structure conformational changes that the macromolecular system undergoes.  In this presentation we discuss the application of smFRET studies to two different systems showing the full potential of this technique to obtain information on both aspects of the problem. As an example of smFRET as a structural tool we show results of experiments on E. Coli Rep helicase. The active form of Rep as a helicase has been shown to be a dimer, but the monomer displays ATP dependent translocase activity. We use eight different Rep mutants site-specific labeled with a fluorescent dye, and a partial duplex DNA substrate that is labeled in one of three possible different locations. The dyes in the protein and in the DNA constitute a donor-acceptor FRET pair giving distance constrains for each labeled site in the protein and each dye location on the DNA in separate experiments.  Through a trilateration process and repeating the experiment in presence of ATP analogs is possible to reconstruct the overall structure of the protein-DNA complex at the different stages of the ATP hydrolysis cycle. To exemplify the use of smFRET for kinetic experiments, we discuss results on the role of T7 helicase in branch migration. It has been shown recently that T7 helicase is able to catalyze branch migration in a 4 way DNA junction (Holliday junction), but little is known about the mechanism involved in this process. Labeling two opposite arms of a Holliday junction with a donor-acceptor pair allows us to follow the branch migration process on real time. Using 4 way junctions with different content of homologous and non-homologous regions we were able to show that unidirectional translocation of the helicase through the duplex region, acting essentially as a molecular rectifier, bias the random walk of the homologous regions favoring the migration in one direction. Because of our few base pair resolution, we can detect single base DNA heterologous regions that appear as stall points in the helicase catalyzed migration reaction and determine the limiting kinetic steps for the overall reaction