Title

The molecular mechanisms of the function of troponin C and troponin I

Date of Award

1992

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmacology

First Committee Member

James D. Potter, Committee Chair

Abstract

The molecular mechanisms of the function of both fast skeletal muscle troponin C (STnC) and troponin I (STnI) have been systematically studied. The functional investigation of TnC thrombin fragments in skinned skeletal muscle fibers confirmed the view that the NH$\sb2$-domain of TnC is essential for triggering muscle contraction, while the -COOH domain is responsible for anchoring TnC to TnI. The entire structure of TnC is necessary to be fully functional. More specific studies of the function of the Ca$\sp{2+}$-specific binding sites of STnC using the mutant with either inactivated site I (VG1) or site II (VG2) showed that both the Ca$\sp{2+}$-specific binding sites of STnC are required for the regulation of muscle contraction. There is a cooperative interaction between sites I and II of STnC. In addition, the Ca$\sp{2+}$-specific binding sites of TnC probably play an important role in determining the differential Sr$\sp{2+}$ sensitivity between skeletal and cardiac muscle contraction. The role of the central helix of TnC in its biological activity was also evaluated by studying the effects of mutations involving amino acid deletion and changes in length and orientation of the central helix. Only the change in the orientation of the two Ca$\sp{2+}$ binding domains appeared to play a major role in affecting TnC activity. To investigate the functional role of the NH$\sb2$-terminus of STnI, a cDNA for rabbit skeletal TnI (RSTnI) was isolated and sequenced. A correction to the originally published amino acid sequence of RSTnC was made, in which Arg-153, Asp-154, and Leu-155 were inserted. Importantly, affinity chromatographic and actomyosin ATPase studies of a deletion mutant of RTnI (TnI$\sb{\rm d57}$), which lacks residues 1-57, provide strong evidence that the NH$\sb2$-terminus of TnI probably constitutes a Ca$\sp{2+}$-Mg$\sp{2+}$ dependent site of interaction with TnC and is also a crucial region for the interaction with TnT. Thus, it appears to play a crucial structural role in stabilizing the Tn complex, while the COOH-terminal half of the molecule has a major regulatory function in actomyosin ATPase activity. Furthermore, from these studies, TnT was demonstrated to confer the Ca$\sp{2+}$ sensitive reversal of actomyosin ATPase activity through direct interaction with TnC.

Keywords

Health Sciences, Pharmacology

Link to Full Text

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