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). These protons are ultimately shuttled through the ATP synthases, which utilise the potential energy of the pmf to catalyse the synthesis of the biological energy carrier, adenosine triphosphate (ATP). ATP synthase is a multi-subunit enzyme complex, and these subunits are organized into two major domains; F/V/A1, the soluble catalytic subunit, and F/V/AO, the transmembrane proton-translocating subunit. To date, all subunits of the F-, V- and A-type ATPases have been structurally characterised, with the exception of the transmembrane proton channel (Figure 1, subunit I) that accepts protons from complex IV of the electron transport chain and delivers them to the membrane rotary ring (Figure 1, subunits K). It is therefore not yet fully understood how the pmf is coupled to ATP synthesis at the atomic 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 structurally characterisation of this subunit has therefore been devised, utilising a number of new 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 will be evaluated by Western blot analysis, and the constructs demonstrating the best expression will be screened further in medium scale expression and affinity purification trials. Preliminary results from small- and medium-scale expression trials indicate that this approach is suitable for this aim. The results of these trials will guide subsequent optimisation strategies, including protein truncation and the introduction of stabilising motifs such as T4 lysozyme into predicted flexible loops. It is anticipated that this approach will ultimately enable the identification of a construct that will enable crystallisation and structure determination by X-ray crystallography of subunit I of the A-type ATP synthase.