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https://hdl.handle.net/2142/35219
Description
Title
Studies of myosin mechanics using fluorescence
Author(s)
Reifenberger, Jeffrey George
Issue Date
2006-10
Doctoral Committee Chair(s)
Selvin, P.
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Molecular Motors
Fluorophores
Lanthanide Probes
Smooth Muscle Myosin
Language
en
Abstract
Molecular fluorescence provides scientists with a wealth of information on how biological systems function at the molecular level. Through fluorescence, individual biological molecules can be tracked as they interact with other proteins, DNA, ligands, etc. and change their conformation in response to the interaction. Molecular motors such as myosin provide an ideal system for fluorescence. The multitude of currently available fluorescent techniques allow for the study of each important step required for myosin to generate directed motion along an actin filament. Several fluorescent techniques such as fluorescence resonance energy transfer (FRET), Lanthanide based resonance energy transfer (LRET), fluorescence imaging with one nanometer accuracy (FIONA), and defocused orientation and position imaging (DOPI) are all applied to specifically myosin II and myosin VI in the text below.
FRET or a modified version known as LRET, allow for the measurement of angstrom level changes in the position between two fluorescent probes attached to a molecule. The advantage of LRET over FRET is that it can measure absolute distances between the two dyes as opposed to only the changes in distance between two different conformations. The anisotropy, a quantity that determines the polarization of emitted light, was measured for several lanthanide probes containing Terbium and Europium and shown to be high for Eu-DTPA (and TTHA), but near zero for Tb-DTPA (or TTHA) and when the lanthanides are excited indirectly through an antenna molecule. The fact that these probes emit un-polarized light results in the high accuracy of LRET. LRET and FRET measured the lever arm swing of smooth muscle myosin II and confirmed that there was an actin dependent state.
FIONA is able to track the position of a single dye with approximately one nanometer accuracy while DOPI is able to determine the dye's orientation. Both of these techniques were used to study the motion of myosin VI and the orientation of its light chain domain (LCD) as the motor walks along actin. Despite conventional wisdom, the LCD of myosin VI did not appear to move as myosin VI took a step. Finally, preliminary single molecule FRET measurements on myosin VI are discussed.
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