Key result
Mutant TnI shows no functional difference from wild-type TnI in regulating acto-S1 ATPase activity.
Why the study?
The functional importance of the three cysteine residues in troponin I was unclear, and a non-oxidizable mutant TnI was needed for structural studies.
Substitution of all three cysteine residues in TnI yields a functional, non-oxidizable mutant suitable for structural studies.
Provides functional non-oxidizable TnI mutant for structural studies; leaves open any clinical translation.
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.
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Kluwe et al. (1993) studied this question. Mutant TnI (cysteine residues substituted) vs. Wild-type authentic TnI was evaluated on Functional differences in interactions with TnT and TnC and regulation of acto-S1 ATPase activity. A mutant troponin I with all three cysteine residues substituted showed no significant functional differences compared to wild-type TnI in regulating acto-S1 ATPase activity.
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