Development of Single Molecule Fluorescence Techniques with Application to Molecular Motors

Download or read book Development of Single Molecule Fluorescence Techniques with Application to Molecular Motors written by Lee Stirling Churchman and published by . This book was released on 2007 with total page 96 pages. Available in PDF, EPUB and Kindle. Book excerpt: FIONA was employed to follow one motor domain of the dimeric myosin VI molecule. We found that myosin VI moves along actin using a "hand-over-hand" mechanism. SHREC was subsequently developed so that both motor domains of dimeric myosin V could be followed simultaneously. Myosin V was also observed to use a "hand-over-hand" mechanism. To study the kinetics of these dimeric myosins working concentrations approaching the Km of ATP binding to the myosin motor domain would be required (∼1 muM). Linear zero-mode waveguides (lZMW) were devised to reduce the background created by high concentrations of labeled ATP. The lZMW's reduce background by evanescent excitation, nanoconfinement, and limited fluorescence coupling out of the waveguide. Their linearity permits actin filaments to be included in the experimental design. As the collection of single molecule datasets employs specialized equipment and is time intensive, data analysis was optimized by statistical analysis to ensure that the most precise information was extracted.