The long-term objective of the proposed work is to understand the molecular basis of the mechanism of muscle contraction. This will involve understanding how ATP hydrolysis is coupled to the large- scale macro-molecular changes responsible for force generation and how the changes are regulated. This work, which has a basic science objective, will generate knowledge which may have implications for cardiac and muscle-related disease. We proposed using neutron solution scattering and neutron helical diffraction to investigate specific structural questions crucial to the understanding of how muscle works. The experiments will utilize the selective deuteration of one, or sometimes two, of the members of the macromolecular complex under study. By varying buffer D2O content, contrast-matching of the buffer with protonated (H) or deuterated (D) protein will be achieved. This will allow examinations of unmatched protein (either H or D) in situ with little or no interference from the other (""""""""invisible"""""""") proteins. Deuterated proteins will be generated by cell biology and recombinant DNA techniques. We will investigate the following questions: (1) What is the in situ structure of regulatory light chains (RLC's) of myosin when the myosin heads are either free in solution or bound to actin with and without Ca2+? What is the separation of RLC's when heads ar bound to actin? (2) What is the in situ structure of troponin-C (TNC) with and without bound Ca2+ and what is the cross-helix radial separation of TNC with and without bound Ca2+? Is the TNC- TNC separation affected by myosin binding to the thin filament? (3) What are the structural changes, if any, that occur in the thin filament when myosin heads bind to actin in the absence of ATP? (4) What is the position and separation of RLC in relaxed, rigor, the putative weak-binding state and in an analog-induced state thought to resemble a force generating acto-myosin intermediate? (5) What is the change in position and intra-myosin separation of light chains when muscles are stretched in rigor? (6) What is the average radial position of the light chain-bearing portion of the myosin head in a muscle during isometric contraction of muscle fibers? Does the intensity of the 14.3 mn meridional reflection (arising from light chains alone) increase during contraction as it does in X-ray diffraction? (7) What are the phases of the equatorial neutron diffraction pattern from rigor vertebrate muscle? Can the low-resolution structure of rigor muscle fibers be determined from neutron diffraction? The answers to these questions will test hypotheses about how muscle contraction and regulation occur.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR039710-03
Application #
3159926
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1989-07-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Mendelson, R; Morris, E P (1997) The structure of the acto-myosin subfragment 1 complex: results of searches using data from electron microscopy and x-ray crystallography. Proc Natl Acad Sci U S A 94:8533-8
Fujiwara, S; Mendelson, R A (1996) In situ shape and distance measurements in neutron scattering and diffraction. Basic Life Sci 64:385-95
Mendelson, R A; Schneider, D K; Stone, D B (1996) Conformations of myosin subfragment 1 ATPase intermediates from neutron and X-ray scattering. J Mol Biol 256:1-7
Stone, D B; Schneider, D K; Huang, Z et al. (1995) The radius of gyration of native and reductively methylated myosin subfragment-1 from neutron scattering. Biophys J 69:767-76
Fujiwara, S; Kull, F J; Sablin, E P et al. (1995) The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study. Biophys J 69:1563-8
Mendelson, R A; Morris, E (1994) The structure of F-actin. Results of global searches using data from electron microscopy and X-ray crystallography. J Mol Biol 240:138-54
Mendelson, R; Morris, E (1994) Combining electron microscopy and X-ray crystallography data to study the structure of F-actin and its implications for thin-filament regulation in muscle. Adv Exp Med Biol 358:13-23
Curmi, P M; Stone, D B; Schneider, D K et al. (1991) Mechanism of force generation studied by neutron scattering. Adv Biophys 27:131-42
Mendelson, R A; Bivin, D; Curmi, P M et al. (1991) Recent neutron scattering studies of muscle contraction and its control. Adv Biophys 27:143-53