1H and 31P 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 ³¹P and ¹H 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 ³¹P 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 ¹H NMR resonances of the tyrosine residue; the amide resonance is shifted -0.02 ppm, the Hα resonance 0.06 ppm, the Hβ resonances 0.10 and -0.04 ppm, the H§ resonances 0.02 ppm and the Hω resonances 0.26 ppm. The pK_a-value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the ³¹P 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 ¹H NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the Hα-resonance -0.43 ppm, the Hβ-resonance 0.03 ppm and the Hγ-resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the Hα-resonance -0.17 ppm, and the Hβ-resonances 0.17 ppm.