The role of glycine (residue 89) in the central helix of EF‐hand protein troponin‐C exposed following amino‐terminal α‐helix deletion

Abstract

Because an N-terminal a-helical (N-helix) arm and a KGK-triplet (residues "KGKgn) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structurefunction correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N-helix and the KGK-triplet in TnC. Noticeably, the amino acid compositions vary between the N-helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGKtriplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant ANt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK9n-triplet (mutant ANtAKGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268: 11685-1 1690). Using this functional test, we find that replacement of Gly-89 with a Leu or an Ala could also overcome the contractile defect associated with N-helix deletion. On the other hand, replacement of the skeletal TnC N-helix with cardiac type N-helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly-89 residue in skeletal TnC and suggest that the N-terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N-helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms.

Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 Å resolution

“…Using a second spectral probe mutant (Phe29Trp; Pearlstone et al, 1992) to measure calcium sensitivity via far and near-UV CD and¯uorescence techniques, these workers showed a reduction of calcium af®nity in the N domain of 1.6 to 1.8-fold and a reduced Ca 2 -activated enzymatic activity in reconstituted troponin-tropomyosinactomyosin S1 and myo®brillar ATPase systems . Similar conclusions have been drawn regarding the critical role of the Nterminal helix in the Ca 2 -mediated activity using a (residues 1 to 14) deletion mutant of avian TnC (Smith et al, 1994) and a (1 to 15) deletion mutant of rabbit skeletal TnC (Ding et al, 1994;Gulati et al, 1993). Our work provides a structural basis for these results.…”

Functional Role of Arginine-11 in the N-Terminal Helix of Skeletal Troponin C: Combined Mutagenesis and Molecular Dynamics Investigation

“…If the force level in the repeat cycle was within 1-2% of that in the first cycle, no further repletion was attempted. The final assurance regarding completion of the loading of the particular protein was derived from quantitative analyses of polyacrylamide gel electrophoresis (SDS-PAGE) developed from selected experimental fiber segments, as described previously (Ding et al. 1994).…”

Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

“…Mutational studies of the central helix in TnC have previously shown that both a required amount of flexibility and a critical length are necessary for optimal activity, suggesting that the linker region may function as a molecular ruler, maintaining a proper relationship between the N-and C-domains of cTnC (35)(36)(37). In the present study, we utilized 15 N R 2 values to measure flexibility within the linker region of cTnC in the presence of full-length cTnI.…”