The F-, V, and A-type ATPases are molecular machines that play a fundamental role in energy conversion in all forms of life. During respiration and photosynthesis, nutrient-derived protons travel through a series of transmembrane complexes to create an electrochemical gradient termed the proton motive force (pmf). The ATP synthases utilise the pmf to catalyse the synthesis of the biological energy carrier, adenosine triphosphate (ATP). ATP synthase is a multi-subunit enzyme complex, with the subunits organised into two major domains; the soluble catalytic F/V/A₁ and the proton-translocating transmembrane F/V/A_O. To date, the only subunit of the F-, V- and A-type ATPases that is yet to be structurally characterised is the transmembrane proton channel (subunit I). It is therefore not yet fully understood how the pmf is coupled to ATP synthesis at the molecular level.

Efforts to purify and crystallise the A-type ATP synthase subunit I from the thermophilic bacterium Thermus thermophilus have thus far proved unsuccessful. A new approach that will enable structural characterisation of this subunit has therefore been devised, utilising a number of high-throughput techniques. To increase the likelihood of obtaining a protein amenable to overexpression, genes encoding subunit I homologues from twelve different species of archaea and bacteria have been cloned into eight different vectors containing numerous combinations of affinity and solubility tags, giving a total of 96 expression constructs. Expression of these constructs has been evaluated by Western blot analysis, and the constructs demonstrating the best expression have been screened further in medium and large scale purification trials. The results of these trials are guiding optimisation strategies, including detergent screens and protein truncation. It is anticipated that this approach will yield a construct that will result in crystallisation and structure determination by X-ray crystallography of subunit I of the A-type ATP synthase.