Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn) , is phosphorylated by the cAMP-dependent protein kinase A at two adjacentl y located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each mo nophosphorylated form cTnI mutants, in which each of the two serine residue s is replaced by an alanine, were generated. These mutants, as well as the wildtype cardiac troponin I (cTnI-WT) have been expressed in Escherichia co li, purified and characterized by isoelectric focusing, MS and CD-spectrosc opy, Monophosphorylation induces conformational changes within cTnI that ar e different from those induced by bisphosphorylation. Functionality was ass essed by measuring the calcium dependence of myosin S1 binding to thin fila ments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosph orylation of cTnI-WT the pCa curve was shifted to the right to the same ext ent as that observed with bisphosphosphorylated native cTnI. However, the a bsolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium aff inity of cTnC: pCa(50)-values were higher than those obtained with the nati ve cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphor ylation has a function different from that of bisphosphorylation; this func tion has not yet been identified.