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 adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.
Reiffert
S
S
Institut für Physiologische Chemie, Universitätsstr, Bochum, Germany.
Maytum
R
R
Geeves
M
M
Lohmann
K
K
Greis
T
T
Blüggel
M
M
Meyer
H E
HE
Heilmeyer
L M
LM
Jaquet
K
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000737:chemistry; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014336:Troponin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020107:Troponin T / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D002118:Calcium