Converting a monomeric arginine kinase to a dimer through directed evolution
There are several possible explanations for why many proteins have evolved higher order tertiary structures. However there is little known about the mechanism of the evolution of oligomers. The phosphogen kinase family is a group of enzymes that are widely distributed throughout the animal kingdom and have diverse quaternary structures making them an ideal model system to study molecular evolution. Arginine kinase (AK) and creatine kinase (CK) are the two prominent members of the phosphogen kinase family and have high sequence homology but differing quaternary structures, existing primarily as a monomer and a dimer respectively. To better understand how oligomers arose in the phosphogen kinase family a novel method for directed evolution was developed in an attempt to convert a monomeric AK into a dimer. Directed evolution is a general term describing a method used to change an enzymeÃs function using selective pressures for an altered function. A selective pressure to form a dimeric AK is generated by triggering the lambda phage repressor system in E. coli. AK was fused with the DNA binding domain of cI lambda phage repressor protein in order to confer immunity to lambda phage by blocking gene expression of several genes required for lambda phage growth. A chemical mutagen facilitated rapid mutagenesis. The starting isoform of AK contained ten parsimonious mutations intended to add residues necessary for dimerization in CK including key residues that would make up the dimer interface. When mutated and infected with lambda phage, several colonies with the AK construct survived. However, sequence data indicated that these survivors were contaminated with a plasmid which conferred immunity to lambda phage. More colonies were observed for E. coli that had been treated with ENU indicating that there might be AK dimers in the background.
© Copyright 2008 Warren C. Swegal