Interaction of a troponin I inhibitory peptide with both domains of troponin C.
Skeletal muscle contraction is regulated by Ca2+ binding to troponin (Tn), a complex of three proteins attached to the actin-tropomyosin filaments. We have been investigating key interactions of the Ca(2+)-binding protein TnC and the inhibitory protein TnI. Previously, we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI, and found that the N-terminal, regulatory domain of TnC formed cross-links to the inhibitory region of TnI (Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J.H. (1990) Biochemistry 29, 299-304). In the present study we have used EDC to form cross-links between TnC and a synthetic peptide, based on residues 104-115 of TnI, which mimics intact TnI in its ability to inhibit actomyosin ATPase activity. Prior to cross-linking, we acetylated the epsilon-amino groups of the nine lysine residues of TnC in order to prevent intramolecular cross-linking. Cross-linked TnC-peptide products were cleaved with CNBr and several proteinases. The resulting cross-linked peptides were purified by HPLC and characterized by amino-acid sequence analysis. Our results indicate that the TnI peptide interacted most strongly with two sites in TnC: Glu-60 and/or Glu-61 in the N-terminal domain, and acidic residue(s) in segment 84-94 of the linker region which connects the N- and C-terminal domains of TnC.
Kobayashi
T
T
Department of Biological Chemistry, School of Medicine, University of Maryland Biotechnology Institute, Baltimore, 21201, USA.
Leavis
P C
PC
Collins
J H
JH
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism; D003488:Cyanogen Bromide; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000737:chemistry; D016296:Mutagenesis; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000378:metabolism; D011817:Rabbits; D014336:Troponin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D019209:Troponin C; D019210:Troponin I; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011725:Pyridines; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; C030953:2-vinylpyridine; D014357:Trypsin; D008239:Lysine; D003488:Cyanogen Bromide; D002118:Calcium