The long-term goal of this proposal is to elucidate the molecular mechanism of troponin-linked calcium regulation of skeletal muscle contraction. Several experimental approaches will be used to gain a detailed understanding of Ca2+-dependent interactions among the following thin filament proteins: the Ca2+-binding troponin C (TnC), the inhibitory troponin I (TnI), actin, and the tropomyosin-binding protein troponin T (TnT).
The specific aims are to elucidate: (1) the mechanism whereby Ca2+ binding to TnC changes its interaction with TnI, thus triggering the series of events leading to muscle contraction; (2) the mechanism whereby Ca2+- dependent changes in TnC-Tnl interactions are transmitted to actin in the thin filament; (3) the vital but little-characterized interactions of TnT with TnC and TnI. The principal experimental methods to be used are: (1) site-directed mutagenesis of the three Tn subunits in order to provide functionally interesting sites for spectroscopic probes and chemical crosslinkers; (2) covalent crosslinking to identify sites of contact among the proteins: (3) frequency domain measurements of fluorescence resonance energy transfer to measure distance distributions between specific sites in thin filament protein complexes: (5) crystallization studies with the goal of preparing troponin subunits and complexes for three-dimensional structure determination by X-ray diffraction. The results of these studies may help to diagnose, prevent, and perhaps ultimately cure, congenital muscle diseases, some resulting from mutations of single amino acids, in which crucial aspects of Ca2+ regulation are not functioning normally.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR041161-02
Application #
2390520
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1996-04-15
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Digel, J; Abugo, O; Kobayashi, T et al. (2001) Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C. Arch Biochem Biophys 387:243-9
Kobayashi, T; Kobayashi, M; Collins, J H (2001) Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I. Biochim Biophys Acta 1549:148-54
Zhao, X; Kobayashi, T; Gryczynski, Z et al. (2000) Calcium-induced flexibility changes in the troponin C-troponin I complex. Biochim Biophys Acta 1479:247-54
Kobayashi, T; Kobayashi, M; Gryczynski, Z et al. (2000) Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments. Biochemistry 39:86-91
Kobayashi, T; Zhao, X; Wade, R et al. (1999) Involvement of conserved, acidic residues in the N-terminal domain of troponin C in calcium-dependent regulation. Biochemistry 38:5386-91
Khaitlina, S; Antropova, O; Kuznetsova, I et al. (1999) Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin. Arch Biochem Biophys 368:105-11