The role of glycine (residue 89) in the central helix of EF-hand protein troponin-C exposed following amino-terminal alpha-helix deletion.
Because an N-terminal alpha-helical (N-helix) arm and a KGK-triplet (residues 88KGK90) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure-function 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 KGK-triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant delta Nt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90-triplet (mutant delta Nt delta KGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685-11690). 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.
Ding
X L
XL
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Akella
A B
AB
Su
H
H
Gulati
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D004492:Edetic Acid / Q000494:pharmacology; D005998:Glycine / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017384:Sequence Deletion; D013696:Temperature; D014336:Troponin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D019209:Troponin C
D011993:Recombinant Fusion Proteins; D014336:Troponin; D019209:Troponin C; D004492:Edetic Acid; D002118:Calcium; D005998:Glycine