Ca2+-induced structural change in the Ca2+/Mg2+ domain of troponin C detected by crosslinking.
Rabbit muscle troponin C was selectively modified at Cys-98 by 1,3-difluoro-4,6-dinitrobenzene. The second function of the bifunctional reagent was triggered at alkaline pH in the presence and absence of Ca2+. The crosslinked troponin C was hydrolyzed by trypsin and the peptides containing a dinitrobenzene moiety were isolated. When troponin C was crosslinked in the presence of Ca2+, the single dinitrobenzene-containing peptide was Gly-89-Arg-100, in which Cys-98 was crosslinked with Lys-90. When crosslinking was performed in the absence of Ca2+, beside the above peptide two additional peptides containing dinitrobenzene were found. One of these peptides is made up of two fragments, Ser-91-Arg-100 and Asn-105-Arg-120, crosslinked between Cys-98 and Tyr-109. The second peptide, Ala-121-Lys-140, contains modified Lys-136, presumably crosslinked with His-135. The data indicate that the distances between the alpha-carbon of Cys-98 and those of Lys-90, Tyr-109, Lys-136 and probably the alpha-carbon distance His-125-Lys-136, do not exceed 14 A. Comparison with the X-ray structure of troponin C (Herzberg, O. and James, M.N.G. (1985) Nature 313, 653-659) indicates that some of the above distances increase on Ca2+-binding.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D055598:Chemical Phenomena; D002621:Chemistry; D002627:Chemistry, Physical; D002845:Chromatography; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D004139:Dinitrofluorobenzene; D014357:Trypsin; C002041:1,5-difluoro-2,4-dinitrobenzene; D008274:Magnesium; D002118:Calcium