The ultimate goal of the proposed research is to understand structure/function relationships that allow myosin to act as a molecular motor, transducing chemical energy into mechanical work during muscle contraction. Efforts in this proposal are focused on elucidating dynamic structural changes at the actin-binding interface of myosin, and correlating these dynamic structural changes with biochemical and mechanical states of the contractile cycle. Fluorescent probes will be introduced at selected sites within the acto-myosin interface using both chemical and photoreactive labelling techniques. The upper 50 kD subdomain of myosin will be photolabelled with methyl coumarin near the 50/20 kD junction and Lys-553 of the lower 50 kD subdomain of myosin will be lableled with fluorescein at Lys-553. A combination of state-of-the-art spectroscopic, biochemical, and mechanical methods will be used to rigorously test current molecular models of muscle contraction. Specifically, the applicant intends to identify the roles of the upper and lower 50 kD subdomains of myosin in the formation of weakly and strongly bound complexes with actin, examine the role of the cleft splitting the 50 kD domain of myosin in mediating the acto- myosin interaction, determine the fraction of actin-attached myosin molecules during isometric contraction and its relationship to muscle fiber stiffness, determine whether or not all strongly bound myosin cross-bridges generate force, and examine how the acto-myosin duty cycle varies with mechanical load.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AR044219-04
Application #
6029992
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lymn, Richard W
Project Start
1996-07-20
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Physiology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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Yengo, Christopher M; Berger, Christopher L (2002) Fluorescence resonance energy transfer in acto-myosin complexes. Results Probl Cell Differ 36:21-30
Yengo, C M; Chrin, L R; Rovner, A S et al. (2000) Tryptophan 512 is sensitive to conformational changes in the rigid relay loop of smooth muscle myosin during the MgATPase cycle. J Biol Chem 275:25481-7
Cooper, W C; Chrin, L R; Berger, C L (2000) Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils. Biophys J 78:1449-57
MacLean, J J; Chrin, L R; Berger, C L (2000) Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states. Biophys J 78:1441-8
Yengo, C M; Chrin, L; Rovner, A S et al. (1999) Intrinsic tryptophan fluorescence identifies specific conformational changes at the actomyosin interface upon actin binding and ADP release. Biochemistry 38:14515-23
Yengo, C M; Fagnant, P M; Chrin, L et al. (1998) Smooth muscle myosin mutants containing a single tryptophan reveal molecular interactions at the actin-binding interface. Proc Natl Acad Sci U S A 95:12944-9
Warshaw, D M; Hayes, E; Gaffney, D et al. (1998) Myosin conformational states determined by single fluorophore polarization. Proc Natl Acad Sci U S A 95:8034-9