Modified calcium-dependent regulatory function of troponin C central helix mutants.
Mutations have been made in the exposed region of the avian troponin C central helix, the D/E linker, which change its length and the orientation of the Ca2(+)-binding domains relative to each other. The region 87Glu-Asp-Ala-Lys-Gly-Lys-Ser-Glu-Glu-Glu97 has been altered in five deletion (d) mutants: dEDA, dKG, dKGK, dSEEE, and dKEDAKGK. The recombinant troponin Cs were expressed in Escherichia coli, purified, and assayed for function. All mutants retained basic troponin C function. They all bound Ca2+ to the low and high affinity sites, and they all were able to confer Ca2+ sensitivity on the regulated actomyosin ATPase. However, the regulatory function of all mutants except dSEEE was defective in one part of the Ca2+ switch or the other. In certain conditions dKGK and dKEDAKGK failed to inhibit fully whereas dEDA and dKG failed to activate the regulated actomyosin ATPase fully. The following general conclusions have been made. (a) The length of the D/E linker per se (assuming the linker is helical) and the orientation of the two Ca2(+)-binding domains relative to each other are not crucial for regulation. (b) The conserved charge cluster 95Glu-Glu-Glu97, in a region of troponin C known to bind to troponin I and postulated to be required for regulation, appears to be unimportant for function. (c) Deletion of 88Glu-Asp-Ala90 resulted in a troponin C that could not activate the actomyosin (or S1) ATPase over the level of actomyosin alone, thus defining a role for troponin C in this aspect of thin filament regulation. The results have been interpreted in terms of the crystallographic structure of troponin C and related to results with analogous calmodulin mutants.
Dobrowolski
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Z
Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Xu
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GQ
Hitchcock-DeGregori
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SE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009218:Myosins / Q000378:metabolism; D009994:Osmolar Concentration; D010641:Phenotype; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000235:genetics* / Q000378:metabolism; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D009218:Myosins; D002118:Calcium