The control of muscle contraction by myosin is studied at the molecular level in molluscan muscles. We have previously shown that in these muscles calcium triggers contraction by binding directly to myosin and that the regulatory components are the regulatory and essential light chains of myosin which maintain the resting state in the absence of calcium. The mechanism of this subunit regulation is most conveniently studied on scallop myosin since the regulatory light chains can be fully and reversibly dissociated from the heavy chains without denaturation. Removal of the RLCs and immobilization of the light chains at a site where the two myosin heads join locks muscle in the """"""""on"""""""" state, losing its ability to relax. The interactions between the light chains and between the heavy and light chains are studied with the aid of photolabile, heterobifunctional cross-linkers, and with chemical and genetic modifications. We plan to identify the cross-linked products formed with various foreign light chains in rest and rigor. Since cross-linkers introduced into the various foreign light chains are located differently in the sequence, the experiments will help to ascertain the polarity, colinearity and relative position of the light chains in myosin in its different functional states. Cross-linkers introduced via the fast reacting heavy chain thiols will relate the heavy chains to light chain movement. Fragmentation of the heavy chain with proteolytic enzymes may identify regions within the molecule responsible for particular functions. Light chains modified by site-directed mutagenesis will help to identify residues responsible for binding to the heavy chain, calcium binding and calcium regulation of contractile functions. Introduction of thiol groups in predetermined positions of the sequence will allow for a more precise localization of the light chains in myosin, and a more accurate description of their rearrangement. The findings will clarify the basic mechanisms of thick-filament regulation They may also be extended to vertebrate smooth and non-muscle myosins, which are also regulated by light chains but triggered by phosphorylation instead of calcium binding.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR015963-29
Application #
2078320
Study Section
Special Emphasis Panel (NSS)
Project Start
1977-05-01
Project End
1997-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
29
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Brandeis University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
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Houdusse, A; Kalabokis, V N; Himmel, D et al. (1999) Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head. Cell 97:459-70
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Roopnarine, O; Szent-Gyorgyi, A G; Thomas, D D (1998) Microsecond rotational dynamics of spin-labeled myosin regulatory light chain induced by relaxation and contraction of scallop muscle. Biochemistry 37:14428-36
Kalabokis, V N; Szent-Gyorgyi, A G (1998) Regulation of scallop myosin by calcium. Cooperativity and the ""off"" state. Adv Exp Med Biol 453:235-40
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Sohn, R L; Vikstrom, K L; Strauss, M et al. (1997) A 29 residue region of the sarcomeric myosin rod is necessary for filament formation. J Mol Biol 266:317-30
Kalabokis, V N; Szent-Gyorgyi, A G (1997) Cooperativity and regulation of scallop myosin and myosin fragments. Biochemistry 36:15834-40

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