Effects of calcium and subunit interactions on surface accessibility of cysteine residues in cardiac troponin

Abstract

Rabbit and bovine cardiac troponin (Tn) subunits and complexes were labeled with iodo-[14C]acetamide in the presence and absence of Ca2+ to determine the effects of tertiary and quaternary structure on exposure of Cys SH groups. This procedure serves both to map regions of subunit interaction and the effects of Ca2+-induced conformational change and to indicate which Cys residues should be useful attachment sites for spectroscopic or cross-linking probes. After being labeled, Tn subunits were purified by using reversed-phase HPLC and subjected to tryptic cleavage with or without prior citraconylation. Cys-containing fragments were isolated by RP-HPLC, and the percent labeling was determined. Cys-75 and -92 of TnI were completely accessible to iodoacetamide both when TnI was labeled alone or when in the TnC-TnI complex. Both residues were largely inaccessible when Tn or the TnI-TnT complex was labeled, suggesting burial in the TnI-TnT interface. In contrast, the Cys from the N-terminal region of bovine TnT was stoichometrically labeled when TnT was labeled alone, in native Tn or in a troponin-tropomyosin complex. Cys-35 and -84 of the TnC are located in the nonfunctional Ca2+ binding loop I of cardiac TnC and helix D, respectively. For TnC alone, the percent labelings of Cys-35 and -84 were 11% and 26%, respectively (minus Ca2+) and 16% and 63%, respectively (plus Ca2+). For TnC alone, the percent labelings of Cys-35 and -84 were 11% and 26%, respectively (minus Ca2+ and 16% and 63%,, respectively (plus Ca2+). For TnC labeled within Tn, the percent labelings of Cys-35 and -84 were 20% and 52%, respectively (minus Ca2+), and 20% and 78%, respectively (plus Ca2+). The Ca2+-induced exposure of these residues, especially Cys-84, supports the Ca2+-activated model of turkey skeletal TnC derived from crystallographic data [Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638].