Myosin is a molecular motor that has
the capacity to convert the energy captured from its hydrolysis
of ATP into mechanical work as it interacts cyclically with actin,
a cytoskeletal protein. The myosin superfamily now consists of at
least 18 different classes of myosin. These myosins use their motile
abilities in a range of biological functions such as muscle contraction,
organelle transport, and cell division. At present, our laboratory
focuses on the class II myosins responsible for muscle contraction
and the class V myosins associated with vesicular transport.
These myosins share significant structural
and functional capacities, i.e. they possess a motor domain that
has hydrolytic, actin-binding, and motor capacities. Emerging from
the motor domain is a light-chain and/or calmodulin-binding domain
that serves as a mechanical lever to amplify small conformational
changes that originate within the motor domain’s active site.
The length of this lever can vary depending on the myosin class.
The differences in both structure and function among the various
myosin classes can provide a model system to help probe the molecular
structure and function of myosin as a chemomechanical enzyme.
Single
Molecule Biophysical Techniques
To characterize the molecular structure
and function of the actomyosin motor, we have developed an array
of single molecule biophysical techniques that can measure the force
and motion of a single molecular motor. Examples of these techniques
are the laser trap,
total internal reflectance fluorescence (TIRF)
microscopy, and microneedle
force assay. Each individual technique or a combination is currently
employed to address interesting research problems ranging from heart
disease to the basic molecular mechanism of motion generation.
Scientific Questions:
Muscle
Contraction: Comparative Molecular Motor Biophysics
Skeletal, smooth, and cardiac muscle
differ substantially in their speeds of shortening. These differences
have been confirmed at the molecular level and relate to the inherent
cycling rate of the different myosin isoforms. In addition, smooth
muscle myosin generates ~4 times the average force of the striated
muscle myosins. How do structural differences between the various
myosin isoforms determine their speeds of movement and force generation?
In collaboration with Dr.
Kathleen Trybus, who expresses mutant smooth muscle myosin in
the Baculovirus/insect cell expression system, structural mutagenesis
is employed to identify domains within the myosin molecule critical
to its molecular mechanics.
Intracellular
Transport
Myosin V is double-headed and carries intracellular cargo by taking
numerous ~36 nm processive steps along its actin track. To be processive,
both heads should have a high duty ratio and be coordinated, so
that forward motion can occur and that at least one head is attached
at any time to prevent the myosin and its cargo from diffusing away
from its actin track. Using the laser
trap and single molecule fluorescence detection techniques,
questions regarding the coordination between heads, what structural
features of the myosin V molecule are necessary for processivity,
and how strain between the heads serves as a 
coordinating
signal are being addressed in collaboration with Dr.
Kathleen Trybus. The movie demonstrates the hand-over-hand mechanism
of myosin V processivity by differentially labeling the heads with
different color quantum dots. The two color images have been offset
by in the y-axis so that the individual quantum dots (i.e. heads)
can be readily distinguished.
Heart
Disease
Single point mutations in cardiac
muscle myosin and actin have been identified as the primary cause
of sudden death in humans afflicted with either Familial Hypertrophic
or Dilated Cardiomyopathy. Since the heart muscle generates the
power needed to support blood flow through the vascular system,
altered power production may result from the mutations to the actomyosin
motor. Therefore, the power generated by mutant cardiac myosin and
actin expressed in humans, transgenic mouse models, or in vitro
are assessed in the force clamp laser trap assay. These studies
will allow assessment of myosin structure: function relationships
at a molecular level and provide a molecular basis for this deadly
disease.
Coupling
Myosin’s Biochemical and Mechanical Cycles
To generate force and motion, myosin
converts chemical energy stored in ATP through a multistep hydrolytic
mechanism. How the various states in its hydrolytic cycle are coupled
to specific mechanical states is far from certain. Therefore, using
fluorescently labeled ATP in the TIRF
microscope combined with the laser
trap, we propose to correlate specific biochemical and mechanical
states of the actomyosin interaction in real time at the level of
a single myosin molecule.
Thin
Filament Regulation
Force generation in striated muscle
results from myosin cyclically interacting with actin, a process
regulated by changes in intracellular Ca2+ and mediated through
the actin-associated regulatory proteins, troponin (Tn) and tropomyosin
(Tm). Early studies suggested that the Ca2+-dependent movement of
Tn-Tm on actin functioned simply as an “on-off” switch,
regulating myosin binding to actin. To understand how Tn-Tm regulates
myosin binding to actin, we study the regulatory process at the
molecular level using reconstituted single thin filaments in the
laser trap assay in collaboration with Dr.
Peter VanBuren.
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