CHARACTERIZATION OF THE CARDIAC TROPONIN-I PHOSPHORYLATION DOMAIN BY P-31 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Cardiac holotroponin can be phosphorylated at serine 23 and/or 24 in t he heart-specific region of bovine troponin I. When isolated freshly i t is composed of a mixture of non-, two mono-, and bisphosphorylated s pecies. At neutral pH the monophosphorylated form carrying phosphate a t serine 24 yields a resonance signal at 4.6 ppm and that carrying pho sphate at serine 23 at 4.4 ppm; the two phosphate groups of the bispho sphorylated form yield only one P-31-NMR signal at 4.2 ppm. From the c hemical shift dependence on pH, pK(a) values have been estimated to be 5.3 and 5.6 for the phosphate groups at serine 24 and serine 23, resp ectively. Both phosphates of the bisphosphorylated form exhibit very s imilar pK(a) values of approximately 5.8. Separation of bisphosphotrop onin I from the complex results in a downfield shift and the appearanc e of two P-31-NMR signals at positions comparable to those of the two monophospho forms, Complex formation of cardiac troponin I with C or T does not alter the spectrum obtained with isolated troponin I; howeve r, the original troponin spectrum is restored by reconstitution of the holocomplex from all three components T, I, and C. Two signals are al so observed with a bisphosphorylated synthetic peptide [PVRRRS(P)S(P)A NYR] respresenting the phosphorylation domain. pK(a) values of about 5 .3 and 5.6 have been determined for serine 7 (corresponding to serine 24 of troponin I) and serine 6 of the peptide (corresponding to serine 23 of troponin I).