H-1 AND P-31 NMR-SPECTROSCOPY OF PHOSPHORYLATED MODEL PEPTIDES

The model peptides glycylglycyltyrosylalanine (Gly-Gly-Tyr-Ala), glycylglycylthreonylalanine (Gly-Gly-Thr-Ala) and glycylglycylserylalanine (Gly-Gly-Ser-Ala) were phosphorylated at the hydroxyl groups of their tyrosyl, threonyl and seryl residues, respectively, and characterized by P-31 and H-1 NMR spectroscopy. The pK(a)-value of the phosphoryl group in the tyrosine-containing peptide determined from the pH dependence of chemical shifts is 5.9, the P-31 chemical shifts at low pH (4.0) and high pH (8.0) are -3.8 and 0.2 ppm, respectively. Phosphorylation also leads to significant shifts of the H-1 NMR resonances of the tyrosine residue; the amide resonance is shifted -0.02 ppm, the H alpha resonance 0.06 ppm, the H beta resonances 0.10 and -0.04 ppm, the H delta resonances 0.02 ppm and the H epsilon resonances 0.26 ppm. The pK(2)-value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the P-31 chemical shifts at low pH (4.0) and high pH (8.0) are -0.1 and 4.8 ppm, respectively. The corresponding values for the serine peptide are 6.1 (pK(a)), 0.6 ppm and 4.9 ppm. Phosphorylation also leads to significant shifts of the H-1 NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the H alpha-resonance -0.43 ppm, the H beta-resonance 0.03 ppm and the H gamma-resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the H alpha-resonance -0.17 ppm, and the H beta-resonances 0.17 ppm. (C) Munksgaard 1994.