A comparative spectroscopic study of tryptophan probes engineered into high- and low-affinity domains of recombinant chicken troponin C.
The spectral properties of three tryptophan-substituted mutants of recombinant chicken troponin C are compared. Site-specific mutagenesis was used to introduce a tryptophan residue into the high-affinity (Ca2+/Mg2+) domain of troponin C at residue position 105, thereby creating the mutant phenylalanine-105 to tryptophan (F105W). The spectral properties of F105W and a double mutant, F29W/F105W, were compared with the mutant phenylalanine-29 to tryptophan (F29W). Since wild-type chicken troponin C does not naturally contain either tyrosine or tryptophan residues, the tryptophan substitutions behaved as site-specific reporters of metal ion binding and conformational change. The residues that occupy positions 29 and 105 are at homologous locations in low-affinity and high-affinity domains, preceding the first liganding residues of binding loops I and III, respectively. Mutant proteins were examined by a combination of absorbance and fluorescence methods. Calcium induced significant changes in the near-UV absorbance spectra, fluorescence emission spectra, and far-UV circular dichroism of all three mutant proteins. Magnesium induced significant changes in the spectral properties of only F105W and F29W/F105W proteins. Tryptophan substitutions allowed Ca(2+)-specific and Ca(2+)/Mg(2+) sites to be titrated independently of one another. Results indicate that there is no interaction between the two binding domains under conditions where troponin C is isolated from the troponin complex. Magnesium-induced changes in the environment of the tryptophan reporter at position 105 were significantly different from those induced by calcium. This suggests that calcium and magnesium differ in their influence on the conformation of the high-affinity, Ca(2+)/Mg(2+) sites.
Trigo-Gonzalez
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Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Racher
K
K
Burtnick
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Borgford
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T
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002645:Chickens; D002942:Circular Dichroism; D004247:DNA; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D014364:Tryptophan / Q000737:chemistry*
D002413:Cations, Divalent; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D014364:Tryptophan; D004247:DNA; D008274:Magnesium; D002118:Calcium