E. coli expression and characterization of a mutant troponin I with the three cysteine residues substituted.
A TnI cDNA was cloned from rabbit fast skeletal muscle, and site-directed mutagenesis was applied to replace all the three cysteine residues, Cys-48 and Cys-64 by Ala and Cys-133 by Ser. The mutant and wild-type TnI were expressed in E. coli and purified to homogeneity. No significant functional differences were observed between the mutant and the authentic TnI in terms of the interactions with TnT and TnC, and the ability of the reassembled Tn complex to regulate the acto-S1 ATPase activity in a calcium-dependent manner. These findings suggest that none of the cysteine residues in TnI are essential for the function of this protein and can be replaced to obtain a non-oxidizable mutant TnI which is much easier to handle and suitable as an alternative to the authentic TnI for various purposes, such as crystallization of TnI and the whole Tn, and 1H NMR studies.
European Molecular Biology Laboratory, DESY, Hamburg, Germany.
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Kluwe
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Maeda
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Maéda
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011817:Rabbits; D014336:Troponin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T
D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D004247:DNA; D000251:Adenosine Triphosphatases; D003545:Cysteine