Scallop myosin is a regulated molecule that is switched on by the direct binding of Ca2+. The globular head portion of myosin (subfragment-1, S1) is the motor that contains the ATPase site and interacts with actin. The regulatory and the essential light chains, which form part of the head, act as inhibitory subunits. Scallop myosin is therefore uniquely suited for studies of the structural states that correspond to rest and activity, for characterizing the architecture of its unusual Ca2+-binding site, and for following light chain rearrangements and other conformational changes that result from Ca2+ binding. We have grown X-ray quality crystals of the 'native' regulatory domain of the scallop myosin head, consisting of a 10 kD fragment of the heavy chain and both light chains, and have obtained an interpretable electron density map of the structure. The atomic-level structure of the native regulatory domain will be determined by X-ray crystallography in the presence and absence of bound Ca2+ ions in order to describe the structure of the Ca2+-binding site, and to identify the sequences responsible for binding the light chains to the heavy chain. The regulatory domain has also been reconstituted from isolated chains and complete function is restored. We will attempt to crystallize and determine the structure of such domains reconstituted with mutant light chains and with light chains derived from both regulated and unregulated myosins. Such studies should clarify the mechanisms of light chain function. Small crystals of S1 have also been obtained, and we will attempt to improve these crystals in order to determine the structure at the atomic level. This result would allow us to describe the Ca2+- and ATP-induced structural changes of the myosin head. Preparation of a regulated S1 will also be attempted. The results of these studies will be applicable to the activation of all regulated myosins (smooth muscle and non-muscle myosins) and will help to clarify the structural changes involved in contraction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR041808-01A1
Application #
2080997
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1994-12-01
Project End
1999-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Brandeis University
Department
Type
Organized Research Units
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
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Himmel, D M; Gourinath, S; Reshetnikova, L et al. (2002) Crystallographic findings on the internally uncoupled and near-rigor states of myosin: further insights into the mechanics of the motor. Proc Natl Acad Sci U S A 99:12645-50
Nyitrai, Miklos; Szent-Gyorgyi, Andrew G; Geeves, Michael A (2002) A kinetic model of the co-operative binding of calcium and ADP to scallop (Argopecten irradians) heavy meromyosin. Biochem J 365:19-30
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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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Houdusse, A; Love, M L; Dominguez, R et al. (1997) Structures of four Ca2+-bound troponin C at 2.0 A resolution: further insights into the Ca2+-switch in the calmodulin superfamily. Structure 5:1695-711
Perreault-Micale, C L; Jancso, A; Szent-Gyorgyi, A G (1996) Essential and regulatory light chains of Placopecten striated and catch muscle myosins. J Muscle Res Cell Motil 17:533-42
Houdusse, A; Silver, M; Cohen, C (1996) A model of Ca(2+)-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. Structure 4:1475-90

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