Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ?.

ABSTRACT: The bacterial ATP synthase (F(O)F(1)) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ?529?kDa), E. coli F(O)F(1) represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F(O)) and a peripheral catalytic complex (F(1)). The ATPase activity of isolated F(1) is fully expressed by the ?(3)?(3)? 'core', whereas single ? and ? subunits are required for structural and functional coupling of E. coli F(1) to F(O). In contrast to mitochondrial F(1)-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F(1)-ATPase catalytic core. Destabilizing the compact conformation of ?'s C-terminal domain with a phosphomimetic mutation (?S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ?3.15?Å resolution.

SUBMITTER: Roy A 

PROVIDER: S-EPMC3490465 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature