Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31-residue deletion at the C-terminus (exon 9).
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.
Bartegi
A
A
Institut National de la Santé et de la Recherche Médicale Unité 249, Université de Montpellier, France.
Ferraz
C
C
Fattoum
A
A
Sri Widada
J
J
Heitz
F
F
Kassab
R
R
Liautard
J P
JP
D000199:Actins; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D009154:Mutation; D009994:Osmolar Concentration; D011817:Rabbits; D014335:Tropomyosin / Q000235:genetics*; D014336:Troponin
D000199:Actins; D014335:Tropomyosin; D014336:Troponin; D004247:DNA; D000251:Adenosine Triphosphatases