CONFORMATION OF THE N-TERMINAL SEGMENT OF A MONOCYSTEINE MUTANT OF TROPONIN-I FROM CARDIAC-MUSCLE

A monocysteine mutant of cardiac muscle troponin I, cTnI(S5C/C81I/C98S ), was generated from a mouse cTnI cDNA clone and expressed in a bacte rial system. Cys-5 was modified with the fluorescent sulfhydryl reagen t IAANS to probe the conformation of the N-terminal extension of the m utant and the mutant complexed with cardiac muscle troponin C, Our emp hasis was on the effect of phosphorylation of Ser-23 and Ser-24 by pro tein kinase A on the conformation of the N-terminal segment. Phosphory lation resulted in an 8-nm red-shift of the emission spectrum of the a ttached IAANS probe and a reduction of its quantum yield by a factor o f 4-5, The intensity decay of nonphosphorylated IAANS-labeled mutant w as complex and had to be described by a sum of three exponential terms , with lifetimes in the range 0.1-5 ns. A fourth component in the rang e 7-9 ns was required to describe the intensity decay of the phosphory lated mutant. Phosphorylation also reduced the weighted mean lifetime, consistent with the changes observed in the steady state fluorescence parameters and a 33% decrease in the global rotational correlation ti me calculated from anisotropy decay data. This change in correlation t ime suggested a decrease in the axial ratio of the protein. The fluore scence changes of the labelled mutant induced by phosphorylation were carried over to its complex with troponin C. The Stern-Volmer plots of acrylamide quenching of the steady-state fluorescence were essentiall y linear for nonphosphorylated mutant but displayed pronounced concave downward curvatures for the phosphorylated protein under all conditio ns phosphorylated cTnI mutant and are consistent with a folded conform ation of the N-tenminal extension induced by phosphorylation of the tw o serines, These conformational changes may play a role in the modulat ion of cardiac muscle contractility by troponin I phosphorylation.