Effects of Ca2+ and subunit interactions on surface accessibility of cysteine residues in cardiac troponin.
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 effect 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 stoichiometrically labeled when TnT was labeled alone, in native Tn or in a troponin-tropomyosin complex. Cys-35 and -84 of 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 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+).(ABSTRACT TRUNCATED AT 250 WORDS)
Ingraham
R H
RH
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Hodges
R S
RS
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D066298:In Vitro Techniques; D007460:Iodoacetamide; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*
D010446:Peptide Fragments; D014336:Troponin; D003545:Cysteine; D002118:Calcium; D007460:Iodoacetamide