Stabilization by a disulfide bond of the N-terminal domain of a mutant troponin C (TnC48/82).
The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the N-terminal domain of rabbit skeletal troponin C (sTnC; Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82 is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide. The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms. Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of TnC.
Gusev
N B
NB
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts.
Grabarek
Z
Z
Gergely
J
J
D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011817:Rabbits; D013696:Temperature; D014336:Troponin / Q000737:chemistry* / Q000235:genetics; D019209:Troponin C; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D014336:Troponin; D019209:Troponin C; D014357:Trypsin