Conserved Asp-137 imparts flexibility to tropomyosin and affects function
John P Sumida1, Eleanor Wu, Sherwin S Lehrer
1Cardiovascular Program, Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
The Journal of Biological Chemistry
|January 1, 2008
Summary
The conserved Asp-137 in tropomyosin (Tm) destabilizes its structure, increasing flexibility. This flexibility is crucial for the cooperative activation of muscle thin filaments by myosin.
Area of Science:
- Muscle physiology
- Protein dynamics
- Biochemistry
Background:
- Tropomyosin (Tm) is an alpha-helical coiled-coil protein essential for muscle contraction.
- Tm regulates the interaction between actin and myosin, controlling muscle force generation.
- Tm's stability is typically maintained by hydrophobic residues, but Asp-137 presents an unusual charged residue.
Purpose of the Study:
- To investigate the role of the conserved Asp-137 in tropomyosin stability and flexibility.
- To determine the impact of Asp-137 on Tm's susceptibility to proteolysis and its regulatory function.
Main Methods:
- Site-directed mutagenesis was used to replace Asp-137 with hydrophobic Leu.
- Limited proteolysis with trypsin was employed to assess Tm susceptibility to cleavage.
- Actin-stabilized Tm and actin-myosin subfragment 1 (S1) ATPase activity were measured.
Main Results:
- The Asp-137 mutant showed significantly reduced susceptibility to trypsin cleavage compared to native Tm.
- Actin binding stabilized Tm, reducing cleavage rates by tenfold.
- The Leu-137 mutant exhibited higher actin-myosin S1 ATPase activity, indicating altered regulatory function.
Conclusions:
- The conserved Asp-137 destabilizes tropomyosin, imparting flexibility to the coiled-coil structure.
- This Tm flexibility is essential for the cooperative activation of the thin filament by myosin.
- A direct link exists between tropomyosin's dynamic properties and its functional role in muscle regulation.
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