Single-Molecule Fluorescence Studies of ReAsH and Myosin VI

119 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.Single-molecule fluorescence spectroscopy has become one of the most powerful techniques in chemistry and biology. Recently, a new fluorescence technique called fluorescence imaging with one nanometer accuracy (FIONA) was shown to localize the position of a fluorescent molecule within a nanometer. Using FIONA, we examined photophysical properties of ReAsH - a novel fluorescent molecule. We showed that ReAsH is more photostable and brighter than eGFP, indicating that ReAsH can be used for in vivo tracking and localization of specific proteins. We also have used FIONA to study myosin VI, the only myosin to move in a direction opposite that of other myosins. This reverse directionality enables myosin VI to be involved in endocytosis. We investigated its structural and functional mechanisms. First, we discovered the stepping mechanism of myosin VI. By placing fluorescent molecules in two different positions and using FIONA, we showed that myosin VI walks hand-over-hand. Second, we revealed the dimerization mechanism of myosin VI. We demonstrated that myosin VI can be dimerized by clustering monomers through binding them either to actin or to antibodies, which implies that cargo-binding induces the dimerization of myosin VI inside cells. Third, we showed that the 53 amino acids unique to myosin VI (the unique insert) determine its directionality. We constructed several chimeric myosins with or without the unique insert, and tested them with gliding assay, which showed that the unique insert causes reverse directionality. Using FIONA, we found that it has a broad distribution similar to that of myosin VI, suggesting that the converter domain is the source of its highly variable step size. Fourth, we measured the processivity of myosin VI at different temperatures, and found that the average step size was smaller at higher temperatures, suggesting that the lever arm becomes less rigid at higher temperatures. Circular dichroism spectrum confirmed a structural change at ∼30°C. We found that additional ADP increases the run-length of myosin VI, implying that the preference of myosin VI for ADP and competition between ADP and ATP for catalytic sites prevents myosin VI from detaching from actin and allows it to walk long distances.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD