Towards the Engineering of an Orthogonal Protein Kinase/Nucleotide Triphosphate Pair

Abstract Remolding protein/ligand interfaces has led to the development of new tools for the study of biological systems. Such methods allow one to engineer proteins with specificity for designed biological probes such as inhibitors, substrates or small molecule dimerizers. Previous work in our laboratory has resulted in engineered protein kinases with specificity for ATP analogs which are otherwise orthogonal ligands for wild type kinases. Kinase reactions of analog-sensitive mutant kinases in cell lysates using γ 32 P labeled ATP analogs allow identification of the direct substrates of the sensitized kinase. As an extension of this methodology, we have designed and evaluated an ATP analog, N 4 (benzyl) ribavirin triphosphate, which may be a suitable phosphodonor for a kinase that does not utilize ATP. Such a modification is likely to be necessary for in vivo kinase substrate labeling experiments where competitive phosphodonors are present in high concentrations.