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 bacterial system. Cys-5 was modified with the fluorescent sulfhydryl reagent IAANS to probe the conformation of the N-terminal extension of the mutant and the mutant complexed with cardiac muscle troponin C. Our emphasis was on the effect of phosphorylation of Ser-23 and Ser-24 by protein kinase A on the conformation of the N-terminal segment. Phosphorylation resulted in an 8-nm red-shift of the emission spectrum of the attached IAANS probe and a reduction of its quantum yield by a factor of 4-5. The intensity decay of nonphosphorylated IAANS-labeled mutant was 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 range 7-9 ns was required to describe the intensity decay of the phosphorylated 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 time calculated from anisotropy decay data. This change in correlation time suggested a decrease in the axial ratio of the protein. The fluorescence changes of the labeled 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 essentially linear for nonphosphorylated mutant but displayed pronounced concave downward curvatures for the phosphorylated protein under all conditions studied. The present results are interpreted in terms of a more compact hydrodynamic shape of the phosphorylated cTnI mutant and are consistent with a folded conformation of the N-terminal extension induced by phosphorylation of the two serines. These conformational changes may play a role in the modulation of cardiac muscle contractility by troponin I phosphorylation.