Phosphorus-31 nuclear magnetic resonance studies of the two phosphoserine residues of hen egg white ovalbumin.
Ovalbumin contains two phosphoserine residues that give rise to two well-resolved resonances in a 31P NMR spectrum. Ovalbumin samples that have been digested with a variety of phosphatases may give rise to only one phosphoserine resonance, indicating that one of the two phosphorylated sites is relatively inaccessible for phosphatase action. By comparison of the amino acid sequence of the peptide containing the nonsusceptible phosphate to the overall primary structure, we have assigned the resonances observed (pH 8.3) at 5.0 and 4.75 ppm to phosphoserines-68 and -344, respectively. pH titration behavior and susceptibility of the phosphoserine residues to phosphatases indicate that both are located on the surface of the protein. Both residues have a pKa = 6.00-6.04. Analysis of the Hill coefficients measured for the pH titrations and the JPH coupling constants indicate that neither residue interacts with other charged groups on the surface of the protein. Frequency dependence of 31P NMR parameters shows that at higher magnetic field strengths the contribution of chemical shift anisotropy to the line width becomes very significant. We have calculated from the field-dependent terms that phosphoserine-344 is mobile with respect to the protein surface but that phosphoserine-68 is more restricted in its motion. The latter is also involved in a pH-dependent conformational change, since it is shielded from hydrolysis by phosphatases at higher pH. A comparison of the amino acid sequence of the phosphoserine-68 site shows that it has a striking homology to the active-site peptides of a wide variety of hydrolytic enzymes. Moreover, a comparison with the primary sequences of casein suggests that both proteins are phosphorylated by a protein kinase that specifically recognizes a Ser-X-Glu peptide.
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009682:Magnetic Resonance Spectroscopy; D010047:Ovalbumin; D010744:Phosphoric Monoester Hydrolases; D010758:Phosphorus; D010768:Phosphoserine; D012694:Serine / Q000031:analogs & derivatives
D015636:Magnesium Chloride; D010768:Phosphoserine; D010758:Phosphorus; D012694:Serine; D010047:Ovalbumin; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium