A. Babu et al., CRITICAL MINIMUM LENGTH OF THE CENTRAL HELIX IN TROPONIN-C FOR THE CA2-CONTRACTION( SWITCH IN MUSCULAR), The Journal of biological chemistry, 268(26), 1993, pp. 19232-19238
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 mul
tiple restriction sequences to facilitate mutagenesis (Babu, A., Su, H
., Ryu, Y. & Gulati, J. (1992) J. Biol. Chem. 267, 15469-15474). Syste
matically, we have deleted 3-12 amino acid residues from the central h
elix and examined their effects in maximally activated skinned muscle
fibers. Limiting the deletions to 7 amino acid residues manifested lit
tle change in maximal force development (Sheng, Z., Francois, J. M., H
itchcock, S. E. & Potter, J. D. (1991) J. Biol. Chem. 266, 5711-5715).
However, with further deletions, we now find that contractility was i
nhibited pari passu; by 12 deletions, the inhibition was complete. The
critical minimum length for the central helix is thereby estimated as
27 angstrom The Ca2+ binding capacity (4 mol of Ca2+/mol of protein)
as well as the structural characteristics (alpha-helicity by CD measur
ements and the fluorescence emitted by Tyr-109) indicated a well prese
rved global conformation of the short mutant. However, surprisingly, t
wo of these short mutants filled each TnC slot under highly specific s
uperloading conditions: one short molecule was taken up in EGTA soluti
on, and the second molecule was captured and retained with Ca2+. They
also rescued the contractile switch, evidently in a bimolecular reacti
on. Another short variant (putative skeletal fast muscle TnC-I-II), in
which the NH2-terminal Ca2+-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 ro
le of the central helix linker in TnC is to keep the two lobes optimal
ly apart, evidently in proximity of their respective target sites on t
roponin I in the fiber.