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https://hdl.handle.net/2142/31324
Description
Title
Studies of myosin mechanics using fluorescence
Author(s)
Reifenberger, Jeffrey George
Issue Date
2006
Doctoral Committee Chair(s)
Selvin, Paul R.
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Molecular fluorescence
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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