PHOSPHORYLATION-INDUCED DISTANCE CHANGE IN A CARDIAC-MUSCLE TROPONIN-I MUTANT

Phosphorylation of two adjacent serine residues in the unique N-termin al extension of cardiac muscle troponin I (cTnI) is known to decrease the Ca2+-sensitivity of cardiac myofilaments, To probe the structural significance of the N-terminal extension, we have constructed two cTnI mutants each containing a single cysteine: (1) a full-length cTnI mut ant (S5C/C81I/C98S) and (2) a truncated cTnI mutant (S9C/C50I/C67S) in which the N-terminal 32 amino acid residues were deleted. We determin ed the apparent binding constants for the complex formation between IA ANS-labeled cardiac troponin C (cTnC) and the two cTnI mutants. The af finities of the cTnC for the truncated cTnI mutant were: (1) 1.5 x 10( 6) M-1 in EGTA, (2) 28.9 x 10(6) M-1 in Mg2+, and (3) 87.5 x 10(6) M-1 in Mg2+ + Ca2+. These binding constants were approximately 1.4-fold s maller than the corresponding values obtained with the full-length cTn I mutant, suggesting a very small contribution of the N-terminal exten sion to the binding of cTnI to cTnC, Cys-5 in the full-length cTnI mut ant was labelled with IAANS, and the distribution of the separation be tween this site and Trp-192 was determined by analysis of the efficien cy of fluorescence resonance energy transfer from Trp-192 to IAANS, Th e following mean distances were obtained with the unphosphorylated ful l-length mutant: 44.4 Angstrom (cTnI alone), 48.3 Angstrom (cTnI + cTn C), 46.3 Angstrom (cTnI + cTnC in Mg2+), and 51.6 Angstrom (cTnI + cTn C in Mg2+ + Ca2+). The corresponding values of the mean distance deter mined with the phosphorylated full-length cTnI mutant were 35.8, 36.6, 34.8, and 37.3 Angstrom. The phosphorylation of cTnI reduced the half -width of the distribution from 9.5 to 3.7 Angstrom. Similar but less pronounced decreases of the half-widths were also observed with the ph osphorylated cTnI complexed with cTnC in different ionic conditions. T hus, phosphorylation of cTnI resulted in a decrease of 9-12 Angstrom i n the mean distance between the sites located at the N- and C-terminal portion of cTnI. Our results indicate that phosphorylation elicits a change in the conformation of cTnI which underlies the basis of the ph osphorylation-induced modulation of cTnI activity.