The goal of this project is to determine three-dimensional structures of several muscle proteins, with special emphasis on those involved in the regulation of muscle contraction, and to relate the resulting structures to physiological events. Tropomyosin and troponin are key components of thin-filament based regulation in skeletal muscle and tropomyosin is also found as a component of actin filaments in a range of smooth muscle and non-muscle cells. We are proposing to complete the structural analysis of tropomyosin to the limit of resolution of the crystals, which is about 3.5 angstroms at present. We will construct an atomic model for the protein, and refine it using restraining methods and/or molecular dynamics. The resulting structure will be related to its role in muscle regulation. We will also determine low resolution structures of complexes of tropomyosin and troponin and fragments of actin in the Bailey crystals at low and high calcium concentrations, to reveal gross changes in the regulatory complex during activation. We are proposing to solve the structure of smooth muscle regulatory complexes, including the tropomyosin-caldesmon complex at low (17 angstrom) resolution to reveal the attachment site of caldesmon on tropomyosin. We will also bind caldesmon fragments containing the putative tropomyosin binding regions of caldesmon in crystals and determine their binding sites on tropomyosin. The results will be compared to those of troponin-containing tropomyosin crystals. We will also determine the locations of the AMP and ATP sites in adenylate kinase, and resulting conformational changes that result on substrate binding, by completing the structure of this enzyme to 2.2 angstroms resolution. The significance of this work is two-fold. The enzyme is a key player in the """"""""energy charge"""""""" of the cell, catalyzing the reaction MgATP + AMP <--> MgADP + ADP. Furthermore, adenylate kinase is a close """"""""relative"""""""" of one domain of the cystic fibrosis gene product, and although not implicated in cystic fibrosis, the study of adenylate kinase may reveal aspects of the function of the cft protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR032764-11
Application #
2078869
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1984-04-01
Project End
1996-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
11
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Rice University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
050299031
City
Houston
State
TX
Country
United States
Zip Code
77005
Berry, Michael B; Bae, Euiyoung; Bilderback, Tim R et al. (2006) Crystal structure of ADP/AMP complex of Escherichia coli adenylate kinase. Proteins 62:555-6
Soman, J; Tao, T; Phillips Jr, G N (1999) Conformational variation of calcium-bound troponin C. Proteins 37:510-1
Berry, M B; Phillips Jr, G N (1998) Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A, Mg2+ Ap5A, and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+. Proteins 32:276-88
Phillips Jr, G N (1997) Structure and dynamics of green fluorescent protein. Curr Opin Struct Biol 7:821-7
Phillips Jr, G N; Chacko, S (1996) Mechanical properties of tropomyosin and implications for muscle regulation. Biopolymers 38:89-95
Hnath, E J; Wang, C L; Huber, P A et al. (1996) Affinity and structure of complexes of tropomyosin and caldesmon domains. Biophys J 71:1920-33
Berry, M B; Meador, B; Bilderback, T et al. (1994) The closed conformation of a highly flexible protein: the structure of E. coli adenylate kinase with bound AMP and AMPPNP. Proteins 19:183-98
Zou, G; Phillips Jr, G N (1994) A cellular automaton model for the regulatory behavior of muscle thin filaments. Biophys J 67:11-28
Avila-Sakar, A J; Schmid, M F; Li, L S et al. (1993) Preliminary electron crystallographic analysis of ice-embedded tropomyosin crystals. J Struct Biol 110:67-74
Chacko, S; Phillips Jr, G N (1992) Diffuse x-ray scattering from tropomyosin crystals. Biophys J 61:1256-66

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