Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C.
Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca2(+)-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-TnI interactions, mainly involving chemically modified proteins or fragments thereof, have led to the widely accepted idea that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) was used to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI. TnC was activated with EDC and N-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of TnI [Grabarek, Z., & Gergely, J. (1988) Biophys. J. 53, 392a]. The resulting cross-linked TnCXI was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linked peptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2(+)-binding site II in the NH2-terminal domain of TnC (residues 46-78) formed cross-links with TnI segments spanning residues 92-167. The most highly cross-linked residues in TnI were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of TnI.
Leszyk
J
J
Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore 21201.
Grabarek
Z
Z
Gergely
J
J
Collins
J H
JH
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D015394:Molecular Structure; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D012697:Serine Endopeptidases / Q000378:metabolism; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008239:Lysine; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide