REGULATION OF CONTRACTION IN MUSCLE AND NON-MUSCLE CELLS

Project Details

Description

The long term goal of the laboratory is to understand mechanisms
of regulation of contraction in muscle and non-muscle cells.
Striated muscle thin filament regulation involving troponin and
tropomyosin is of particular interest. Experiments are outlined in
the present proposal to use site-directed mutagenesis for study of
structure-function relationships in troponin C. Troponin C binds
calcium and regulates contraction via interaction with troponin I
and troponin T. Crystallographic analysis of the structure has
shown that troponin C, like calmodulin, has two calcium binding
domains connected by a long central helix. The structural
features of the protein that are important for its function as a
calcium binding regulatory protein and transmission of
information about the state of calcium binding in the regulatory
domain to other components of the thin filament will be
investigated. A cDNA encoding avian troponin C has been constructed from
oligonucleotides and the protein has been expressed and purified
from E. coli. The functional role of the long helix connecting the
two domains of troponin C, a conserved structure in calcium
regulatory proteins, will be evaluated by altering its structure in a
variety of ways: its helicity and stability, its length and
orientation of the domains relative to each other.
Oligonucleotide directed mutagenesis will be used to change
individual residues and to make deletions and insertions. The
mutants will be analyzed for calcium binding, complex formation
with other troponin components, regulation of the actomyosin
ATPase, and ultimately substitution in skinned fibers and X-ray
crystallography. An additional aim of the experiments is to
contribute to our general understanding of the requirements for
alpha-helical structure in proteins. The second specific aim is to determine the location and
structural requirements of the troponin I and troponin T binding
sites on-troponin C. We will use two approaches: construction of
troponin C-calmodulin chimeras and mutagenesis of residues on
the surface of the protein that do not contribute to the
hydrophobic core of the protein. Analysis of chimeras should
indicate which residues are critical for troponin C specific
functions. Mutagenesis of specific residues will follow.
StatusFinished
Effective start/end date12/31/896/30/07

Funding

  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $333,483.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $279,123.00
  • National Institute of General Medical Sciences: $341,508.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $341,508.00
  • National Institute of General Medical Sciences: $295,928.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $341,508.00
  • National Institute of General Medical Sciences: $287,400.00
  • National Institute of General Medical Sciences

ASJC

  • Cardiology and Cardiovascular Medicine
  • Radiation
  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Structural Biology
  • Spectroscopy
  • Cell Biology

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