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.
Dong
W J
WJ
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294-2041, USA.
Chandra
M
M
Xing
J
J
Solaro
R J
RJ
Cheung
H C
HC
D000818:Animals; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003545:Cysteine; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D051379:Mice; D009154:Mutation; D009206:Myocardium / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry*; D010768:Phosphoserine / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D019209:Troponin C; D019210:Troponin I; D010768:Phosphoserine; C011488:IAANS; D017868:Cyclic AMP-Dependent Protein Kinases; D003545:Cysteine