Reiffert S, Maytum R, Geeves M, Lohmann K, Greis T, Blüggel M, Meyer H E, Heilmeyer L M, Jaquet K
Institut für Physiologische Chemie, Universitätsstr, Bochum, Germany.
Eur J Biochem. 1999 Apr;261(1):40-7. doi: 10.1046/j.1432-1327.1999.00261.x.
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.
心肌肌钙蛋白I（cTnI）是心肌肌钙蛋白（cTn）的抑制亚基，在心脏特异性N端延伸区内两个相邻的丝氨酸残基处被依赖cAMP的蛋白激酶A磷酸化。可形成四种不同的磷酸化状态。为了研究每种单磷酸化形式的cTnI突变体，构建了两个丝氨酸残基分别被丙氨酸取代的突变体。这些突变体以及野生型心肌肌钙蛋白I（cTnI-WT）已在大肠杆菌中表达、纯化，并通过等电聚焦、质谱和圆二色光谱进行了表征。单磷酸化诱导的cTnI构象变化不同于双磷酸化诱导的变化。通过测量肌球蛋白S1与含有重组天然、野生型和突变体cTn复合物的细肌丝结合的钙依赖性来评估功能。在所有情况下都获得了功能性的全肌钙蛋白复合物。cTnI-WT双磷酸化后，pCa曲线向右移动的程度与双磷酸化天然cTnI观察到的相同。然而，当使用重组cTn亚基进行重组时，中点的绝对值更高。重组本身改变了cTnC的钙亲和力：pCa50值高于天然心肌全肌钙蛋白复合物的pCa50值。显然，只有cTnI的双磷酸化会影响细肌丝的钙敏感性，因此单磷酸化具有与双磷酸化不同的功能；该功能尚未确定。
