Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C.
We have used the sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) to study the interaction of rabbit skeletal muscle troponin C (TnC) and troponin I (TnI). TnC was specifically labeled at Cys-98 by the maleimide moiety of BPMal, and a binary complex was formed with TnI in the presence of Ca2+. Upon photolysis, covalent cross-links were formed between TnC and TnI [Tao, T., Scheiner, C.J., & Lamkin, M. (1986) Biochemistry 25, 7633-7639]. The cross-linked heterodimer was digested with cyanogen bromide, pepsin, and chymotrypsin into progressively smaller cross-linked peptides, which were purified by HPLC and then characterized by amino acid analysis and sequencing. We obtained a fraction from the initial CNBr digest that contained the expected peptide CB9 (residues 84-135) of TnC, cross-linked mainly to CN4 (residues 96-116), the "inhibitory region" of TnI. The peptides CN1 and CN3 of TnI were also detected in this fraction, but their molar ratios (compared to CB9) were only about 0.15 each, compared to 0.60 for CN4. Sequence analyses of fractions obtained after peptic and chymotryptic digests of the cross-linked CNBr fraction confirmed that CB9 and CN4 were the major cross-linked species. Quantitative analysis of sequencer results indicated that the residues in TnI that appeared to be most highly cross-linked to Cys-98 of TnC were Arg-108 and Pro-110, and to a lesser extent Arg-103 and Lys-107. These findings are consistent with previous studies on interactions between TnI and TnC and provide, for the first time, direct information on the identities of proximate amino acids in the two proteins.
Leszyk
J
J
Department of Biology, Clarkson University, Potsdam, New York 13676.
Collins
J H
JH
Leavis
P C
PC
Tao
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003488:Cyanogen Bromide; D008301:Maleimides; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D001577:Benzophenones; D003432:Cross-Linking Reagents; D008301:Maleimides; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; C046109:benzophenone-4-maleimide; D002918:Chymotrypsin; D003488:Cyanogen Bromide