Analysis of the Oligomeric State of the Mating ATPase ConE and its Arginine Finger Mutant

Abstract

During conjugation, mating pores allow bacteria to transfer DNA to recipient cells. ConE is one of several proteins that make up the mating pore of Bacillus subtilis. ConE, and its‐characterized homologs VirB4 and TrwK, belongs to the FtsK‐HerA superfamily of ATPases which contain a conserved arginine finger that is a good predictor for ring formation and inter‐protomer interactions. Several superfamily members have been purified and characterized in vitro; they form monomers, dimers, and higher order oligomers in solution and generally form hexamers when crystallized. We tested if ConE oligomerizes and how the mutation of ConE's arginine finger affects its oligomerization. His6‐ConE and mutant His6‐ConE (R726A) were purified via nickel affinity chromatography. Purification was monitored via SDS‐PAGE and multimeric states were determined by BN‐PAGE. We found that both wild type and mutant His6‐ConE form monomers, dimers, and higher oligomers. Addition of ATP, single‐stranded DNA, and double‐stranded DNA did not dramatically alter the oligomeric states.