Critical minimum length of the central helix in troponin C for the Ca2+ switch in muscular contraction.
In the troponin C (TnC) dumbbell, the NH2- and COOH-terminal lobes are well delineated, but the role of the central helix and especially the function of its long length remain doubtful. To study this, we used a cDNA construct encoding rabbit fast-twitch muscle TnC, comprising multiple restriction sequences to facilitate mutagenesis (Babu, A., Su, H., Ryu, Y. & Gulati, J. (1992) J. Biol. Chem. 267, 15469-15474). Systematically, we have deleted 3-12 amino acid residues from the central helix and examined their effects in maximally activated skinned muscle fibers. Limiting the deletions to 7 amino acid residues manifested little change in maximal force development (Sheng, Z., Francois, J. M., Hitchcock, S. E. & Potter, J. D. (1991) J. Biol. Chem. 266, 5711-5715). However, with further deletions, we now find that contractility was inhibited pari passu; by 12 deletions, the inhibition was complete. The critical minimum length for the central helix is thereby estimated as 27 A. The Ca2+ binding capacity (4 mol of Ca2+/mol of protein) as well as the structural characteristics (alpha-helicity by CD measurements and the fluorescence emitted by Tyr-109) indicated a well preserved global conformation of the short mutant. However, surprisingly, two of these short mutants filled each TnC slot under highly specific superloading conditions: one short molecule was taken up in EGTA solution, and the second molecule was captured and retained with Ca2+. They also rescued the contractile switch, evidently in a bimolecular reaction. Another short variant (putative skeletal fast muscle TnC-I-II), in which the NH2-terminal Ca(2+)-binding sites were incapacitated, failed to respond to superloading, indicating that sites III and IV could not substitute for sites I and II. The results suggest that a critical role of the central helix linker in TnC is to keep the two lobes optimally apart, evidently in proximity of their respective target sites on troponin I in the fiber.
Babu
A
A
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Rao
V G
VG
Su
H
H
Gulati
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008961:Models, Structural; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009132:Muscles / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D004533:Egtazic Acid; D004247:DNA; D002118:Calcium