A fluorescence polarization based binding assay for Protein Arginine Methyltransferases

Protein Arginine Methyltransferases (PRMTs) are a family of enzymes which function in cellular signaling by modifying other proteins. The PRMTs are involved in a number of signaling cascades which control crucial processes such as the cell cycle, DNA repair, and gene expression. Dysfunction of these enzymes has been implicated in several serious diseases which include heart disease and cancer. The PRMTs work by catalyzing the transfer of a methyl group from the methyl donor S-adenosylmethionine (AdoMet) to a terminal nitrogen of an arginine residue in the substrate protein. Depending on the degree of methylation and the particular PRMTs involved, the PRMT family is capable of generating three distinct species of methylarginine on substrate proteins. Arginine methylation has a number of different consequences depending on the species of methylarginine produced, and the identity of the modified protein. However it is not well understood how the PRMTs are coordinated to deposit the appropriate methyl mark on the appropriate substrate at the appropriate time. Understanding the factors that influence the interactions between the PRMTs and their substrates will be required to understand the mechanisms that control arginine methylation. We are currently working to develop an in vitro fluorescence polarization based competition binding assay to assess the strength of the interaction between various PRMTs and their substrates. When a population of fluorescently labeled solutes are excited by polarized light, the emitted light is partially depolarized. The degree to which the emitted light is depolarized is dependent on the size of the solute. Since the binding of fluorescently labeled substrate to a PRMT effectively changes the size of the solute, this binding can be quantified by measuring the change in fluorescence polarization. Exploiting this property will allow us to assess how strongly the different PRMTs interact with various substrates under different conditions