Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31‐residue deletion at the C‐terminus (exon 9)
- 1 December 1990
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 194 (3), 845-852
- https://doi.org/10.1111/j.1432-1033.1990.tb19478.x
Abstract
The cDNA coding for human skeletal muscle beta-tropomyosin was expressed in Escherichia coli to produce an unacetylated beta-tropomyosin. This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E. coli to produce an unacetylated beta-tropomyosin mutant lacking the C-terminal residues 254-284. The main structural and functional properties of the two isolated proteins, designated tropomyosin-1 and des-(254-284)-tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle alpha beta-tropomyosin. The folding and thermal stability of the three tropomyosins were indistinguishable. Tropomyosin-1, but not des-(254-284)-tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex. Despite its weak binding to actin, des-(254-284)-tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment-1 ATPase in the presence of Ca2+ and low concentrations of subfragment-1. The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament.Keywords
This publication has 36 references indexed in Scilit:
- Structural analysis of human skeletal β-tropomyosin produced in E. coliBiochimie, 1989
- Evolution of tropomyosin functional domains: Differential splicing and genomic constraintsJournal of Molecular Evolution, 1988
- Tropomyosin crystal structure and muscle regulationJournal of Molecular Biology, 1986
- .alpha.-Helix to random-coil transition of two-chain, coiled coils: experiments on the thermal denaturation of doubly crosslinked dimeric .beta.-tropomyosinBiochemistry, 1986
- Removal of tropomyosin overlap and the co-operative response to increasing calcium concentrations of the acto-subfragment-1 ATPaseJournal of Molecular Biology, 1985
- Structural interpretation of the two-site binding of troponin on the muscle thin filamentJournal of Molecular Biology, 1981
- Equilibrium of the actin-tropomyosin interactionJournal of Molecular Biology, 1979
- Tropomyosin coiled-coil interactions: Evidence for an unstaggered structureJournal of Molecular Biology, 1975
- Structural role of tropomyosin in muscle regulation: Analysis of the X-ray diffraction patterns from relaxed and contracting musclesJournal of Molecular Biology, 1973
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970