Probing for structural motion and cooperativity during the PRMT catalytic cycle

Protein arginine methyltransferases (PRMTs) are homodimeric enzymes which are responsible for regulating the expression of genes and the activity of other proteins in humans and other organisms, by transferring a methyl group from S-adenosyl-methionine (SAM) to the terminal guanidino nitrogen atoms of protein arginine residues. PRMT1 is the major producer of methylated arginine in the human body. The homodimer of PRMT1 possess two active sites, each of which appear identical in crystal structures and are therefore both likely able to methylate substrates. Both active sites are obstructed by an N-terminal helical region of each respective monomer, which likely regulates the binding of both the methyl donor SAM, and the arginine containing peptide substrate. In order to observe both the motion in the N-terminal region and observe any potential cooperativity between monomers we would like to be able to selectively label only one N-terminus within a dimer, or knock out activity in one active site. We will achieve this by creating a single polypeptide harboring the sequence of two PRMT1 monomers connected by a short amino acid linker. This linked-PRMT construct (PRMT1-linker-PRMT1) will allow us to selectively label desired positions on only one part of a monomer, such as an active site, along with mutating or truncating one N-terminus at a time. This new construct will allow us to place a fluorophore at only one N-terminus so that we can observe the motion at one active site, while specific mutations or truncations can give us insight into the regulatory role of particular residues or interactions. These new constructs will also allow us to make point mutations in one active site of the dimeric enzyme in order to evaluate the suspected dynamics and cooperativity that occur in PRMT1