The molecular recognition of phosphorylated proteins by designed polypeptides conjugated to a small molecule that binds phosphate

The conjugation of polypeptides from a designed set to the small molecule ligand 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, which in the presence of Zn2+ ions binds inorganic phosphate, has been shown to provide a polypeptide conjugate that binds alpha-casein, a multiply phosphorylated protein, with a dissociation constant K-D of 17 nM. The measured affinity is more than three orders of magnitude higher than that of the small molecule ligand for phosphate and the binding of 500 nM of alpha-casein was not inhibited by 10 mM phosphate buffer, providing a 2000-fold excess of phosphate ion over protein. The selectivity for phosphoproteins was demonstrated by extraction of alpha-casein from solutions of various complexity, including milk and human serum spiked with alpha-casein. In addition to alpha-casein, beta-casein was also recognized but not ovoalbumin. Conjugation of a polypeptide to the zinc chelating ligand was therefore shown to give rise to dramatically increased affinity and also increased selectivity. A set of polypeptide conjugates is expected to be able to capture a large number of phosphorylated proteins, perhaps all, and in combination with electrophoresis or mass spectrometry become a powerful tool for the monitoring of phosphorylation levels. The presented binder can easily be attached to various types of surfaces; here demonstrated for the case of polystyrene particles. The example of phosphoproteins was selected since posttranslational phosphorylation is of fundamental importance in cell biology due to its role in signaling and therefore of great interest in drug development. The reported concept for binder development is, however, quite general and high-affinity binders can conveniently be developed for a variety of proteins including those with posttranslational modifications for which small molecule recognition elements are available.