MOLECULAR ANGLE TRANSITIONS IN MUSCLE

  • Burghardt, Thomas P, (PI)

Project: Research project

Project Details

Description

The cyclic interaction of myosin and actin are at the heart of the mechanism
of muscle contraction and cell motility. A model of the molecular mechanism
of muscle contraction has not been definitively confirmed. The purpose of
the proposed experiments is to elucidate this mechanism by detailed studies
of protein orientation and conformation. The interaction of myosin and
actin will be investigated using static and time resolved techniques
developed for fluorescence and electron spin resonance (ESR) probes. These
probes will be covalently and specifically attached primarily to two myosin
sulfhydryl side chains in muscle fibers, either SH1 or SH2. The probes will
report their orientation as a function of time and physiological state of
the fiber. We will use novel ESR and fluorescence techniques to investigate
the static and time resolved angular distribution of probes attached to the
components of the contractile apparatus. In the static experiments, we
investigate the orientation of the cross-bridge using fluorescence
polarization spectroscopy. With FPS the orientation of the transition
dipole of the probe rotates on the cross-bridge, due to excitation
wavelength variation, allowing a more general study of the cross-bridge
angular degrees of freedom. With ESR the probe angular distribution is
reconstructed, with high angular resolution, from a series of ESR spectra
originating from a muscle fiber at differing tilt angles relative to the
Zeeman magnetic field. The fluorescence polarization and ESR spectra will
be measured from labeled muscle fibers in a variety of physiological states
to determine the cross-bridge orientation in these states and to correlate
this data with the models for cross-bridge participation in muscle
contraction. In the time-resolved experiments, we will investigate the
rotational motions of cross-bridges during muscle contraction on the time
domain of 10-6 to 10+3 seconds. This will be done using the technique of
polarized fluorescence photobleaching recovery (PFPR) applied to labeled
muscle fibers. With PFPR a brief pulse of polarized focused light
irreversibly photobleaches probes with transition dipoles aligned with the
polarization of the light in the focused spot. The bleached region is
monitored with an attenuated polarized light source and the recovery of
fluorescence is observed as unbleached fluorophores rotate and their dipoles
move into alignment with the attenuated polarized source.
StatusFinished
Effective start/end date1/1/906/30/03

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $220,600.00
  • National Institutes of Health: $227,218.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $221,673.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $220,600.00
  • National Institutes of Health

ASJC

  • Medicine(all)

Fingerprint Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.