PROJECTS
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Structural biology of muscle
contraction and its regulation.
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Electron microscopy and x-ray
diffraction of striated muscle.
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Structure of contractile proteins
and their assembly.
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Molecular mechanism of muscle
contraction and its regulation.
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Thick filament structure: surface
arrangement of myosin heads, packing of myosin tails to form the backbone.
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Thick filament function: molecular
mechanism of phosphorylation, control of activation.
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Our current research project
will elucidate the structure of myosin thick filaments from striated muscle
and the structural mechanism of how are they activated by calcium. Our
work will reveal the near atomic structure of the arrangement of myosin
heads on the surface of thick filaments. This will be achieved by determining
the molecular structure of thick filaments using cryo-electron microscopy
and atomic force microscopy; and fitting the atomic coordinates of the
myosin head onto their envelopes.
Our specific objectives
are:
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(1) to accomplish a near-atomic
description of the helical arrangement of myosin heads on the thick filament
surface, revealing the conformation and interactions between myosin heads
which produces the relaxed state of myosin filaments.
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(2) to unravel the structural
basis of how myosin heads helices are established and maintained in the
presence of ATP, by capturing the structural changes that occur when myosin
helices are formed.
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(3) to disclose the molecular
details of the myosin-linked regulation that occurs through the mechanism
of phosphorylation of the regulatory ligth chains of myosin, by capturing
the structural changes that occur when myosin filaments are activated.
Our preliminary results
indicate that these objectives are achievable. I will use two complementary
structural techniques:
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(1) cryo-electron microscopy,
which enables observing specimen molecular structure close to its native
state (frozen-hydrated) at about 1nm resolution avoiding usual EM artifacts;
and through rapid freezing, capturing dynamic events. Three-dimensional
surface of thick filaments will be revealed by computing 3d maps by the
3d-helical reconstruction technique. Isolated thick filaments from striated
muscle will be rapidly frozen and observed unstained in the frozen-hydrated
state. Arresting of transient molecular conformations will be done by using
the spray-freezing technique, to induce fast increases of either ATP or
Ca2+ levels.
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(2) atomic force microscopy,
which enables visualizing directly the thick filament surface but at its
physiological temperature and aqueous liquid environment as compared with
very low temperature and vitreous frozen-hydrated state essential for cryo-EM.
As different than cryo-EM, AFM permits re-scanning of the thick filament
specimen, opening to us the possibility of physiological experiments. By
combining both structural observations (cryo-EM and AFM) with the available
atomic coordinates of the myosin head, we will approach a near-atomic description
of two early stages of the contractile event: the relaxed state and the
activated state, by defining more precisely the location of the domains,
light chains and nucleotide-binding site of the myosin head.
This project will contribute
to a better understanding of the structure of the relaxed thick filament
from striated muscle and how it is activated by calcium. This information
will be important for deciphering the initial stages of the molecular mechanism
of muscle contraction, which could be relevant for the understanding of
more general cell motility molecular mechanisms based on similar arrays
of myosin molecules.
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