Regulation of muscle contraction is difficult to study because it involves changes in several proteins that are in a highly organized system. Biochemical studies provide detailed information of the components but at the loss of the organization. Fiber studies preserve the structure but give little information about the function of the components. By exchanging native regulatory proteins in a single fiber with proteins containing a probe, it is possible to study the function of single components within a fiber. Fluorescent probes on troponin and tropomyosin have been shown, in solution studies, to be sensitive to calcium binding or to the fraction of actin in the active configuration. When placed into single fibers, the fluorescent probes can be used to monitor the state of the regulatory apparatus during various mechanical perturbations. This allows one to follow individual regulatory proteins in the organized structure of the fiber and to answer many questions related to regulation. The method of exchange that is used is an improvement over older methods and leaves the mechanical properties of the fibers unchanged. The plan is to study the exchange process so that it may be optimized and extended it to other proteins in striated muscle. A number of fluorescent probes will be placed on different regions of the troponin complex and the fluorescence changes that occur in fibers will be correlated with those occurring in solution under controlled conditions. In addition to the introduction of fluorescent probes, the exchange process will be used to introduce other well characterized modified or mutant troponins into fibers to study the function of particular regions of troponin. The intent is to begin by examining the effect of the mutations of the regulatory apparatus found in patients with hypertrophic cardiomyopathy. This work is part of a collaborative effort with Dr. Bernhard Brenner (mechanical studies), Dr. Leepo C. Yu (structural studies) and Dr. Yi-der Chen (mathematical modeling).

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR044504-02
Application #
2700234
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1997-05-01
Project End
2000-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
East Carolina University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Greenville
State
NC
Country
United States
Zip Code
27858
Varughese, Jayson F; Baxley, Tamatha; Chalovich, Joseph M et al. (2011) A computational and experimental approach to investigate bepridil binding with cardiac troponin. J Phys Chem B 115:2392-400
Mathur, Mohit C; Chase, P Bryant; Chalovich, Joseph M (2011) Several cardiomyopathy causing mutations on tropomyosin either destabilize the active state of actomyosin or alter the binding properties of tropomyosin. Biochem Biophys Res Commun 406:74-8
Varughese, Jayson F; Chalovich, Joseph M; Li, Yumin (2010) Molecular dynamics studies on troponin (TnI-TnT-TnC) complexes: insight into the regulation of muscle contraction. J Biomol Struct Dyn 28:159-74
Mathur, Mohit C; Kobayashi, Tomoyoshi; Chalovich, Joseph M (2009) Some cardiomyopathy-causing troponin I mutations stabilize a functional intermediate actin state. Biophys J 96:2237-44
Mathur, Mohit C; Kobayashi, Tomoyoshi; Chalovich, Joseph M (2008) Negative charges at protein kinase C sites of troponin I stabilize the inactive state of actin. Biophys J 94:542-9
Wirth, A; Schroeter, M; Kock-Hauser, C et al. (2003) Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1. J Physiol 549:489-500
She, M; Trimble, D; Yu, L C et al. (2000) Factors contributing to troponin exchange in myofibrils and in solution. J Muscle Res Cell Motil 21:737-45
Brenner, B; Kraft, T; Yu, LC et al. (1999) Thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle Biophys J 77:2677-91
Brenner, B; Chalovich, JM (1999) Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contra Biophys J 77:2692-708
Xu, S; Malinchik, S; Frisbie, S et al. (1998) X-ray diffraction studies of the cross-bridge intermediate states. Adv Exp Med Biol 453:271-8;discussion 278-9

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