Synthesis of a troponin C cDNA and expression of wild-type and mutant proteins in Escherichia coli.
An avian fast striated muscle troponin C cDNA was designed and synthesized from six oligonucleotides using the overlap-fill in method and overproduced in Escherichia coli for the purpose of developing recombinant DNA approaches to study structure-function relationships in this calcium-binding regulatory protein. The recombinant protein isolated from E. coli functions as a bona fide troponin C in all properties that were assayed: calcium binding, calcium-dependent conformational change, calcium-dependent interaction with troponin I, and formation of a functional ternary complex with troponin I and troponin T that can confer calcium sensitivity on the actomyosin MgATPase. The initiating methionine was removed by E. coli leaving alanine as the first amino acid, as in the muscle troponin C. The first amino acid was not acetylated, but this difference from the muscle protein has no apparent effect on the function. The presence of Glu at position 99, as in turkey, versus Ala in chicken resulted in no detectable difference in comparing recombinant with chicken troponin C. A mutant in which residues 91-93 (Lys-Gly-Lys) in the D/E helical linker were deleted differs in function from wild-type troponin C in the conformational change that takes place upon calcium binding and its interaction with troponin I. Also, the mutant troponin C is impaired in its ability to form a functional complex with troponin I and troponin T that will confer calcium sensitivity on the actomyosin MgATPase.
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Department of Anatomy, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Hitchcock-DeGregori
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D000205:Actomyosin / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003062:Codon; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics* / Q000502:physiology; D013329:Structure-Activity Relationship; D014336:Troponin / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D019209:Troponin C; D019210:Troponin I
D003062:Codon; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D004247:DNA; D000205:Actomyosin; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium