Clustering of phosphorylated amino acid residues in neurofilament proteins as revealed by phosphorus-31 NMR

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Abstract
The state of phosphorylation in neurofilament (NF) proteins is studied by the 31P NMR technique. The 31P NMR spectrum of intact NF proteins at pH 7.0 is comprised of a major resonance at 4.18 ppm and a minor resonance at 3.53 ppm. The chemical shifts of the major and minor resonances are strongly dependent on pH and have pKa values for phosphoserine of 5.85 and for phosphothreonine of 6.00, respectively. 31P NMR spectra of isolated NF polypeptides show nonequivalent phosphoserine clusters in NF150 and in NF200. Their chemical shifts are very similar in both polypeptides, but the intensities of homologous resonances are different. NF68 has no detectable 31P resonance signal. Phosphate-specific monoclonal antibodies to NF200 can distinguish phosphates of various clusters. Microtubule proteins can also produce specific alteration of the 31P resonances of NF200. NF proteins digested by calcium-activated neutral protease (CANP) show relatively little change in 31P resonances.

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