A high‐throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase ‘inserted’ state

We developed a large scale in vivo protein photocrosslinking analysis pipeline based on the introduction of unnatural amino acid into target protein via scarless genome‐targeted site‐directed mutagenesis, and probing cross‐linked products via high‐throughput polyacrylamide gel electrophoresis. We uncovered that bacterial ATP synthase exists as an equilibrium between the ‘inserted’ and ‘noninserted’ state, maintaining a proper level of net ATP synthesis when shifting to the former under unfavorable energetically stressful conditions. ATP synthase, a highly conserved protein complex that has a subunit composition of α3β3γδεab2c8–15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F1 sector (α3β3γδε) and a membrane‐integrated Fo sector (ab2c8–15). Structural analyses of the isolated protein components revealed that, remarkably, the C‐terminal domain of its ε‐subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown. In an attempt to decipher this, we developed a high‐throughput in vivo protein photo‐cross‐linking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome‐targeted site‐directed mutagenesis technique, and probing the cross‐linked products via the high‐throughput polyacrylamide gel electrophoresis technique. Employing this pipeline, we examined the interactions involving the C‐terminal helix of the ε‐subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the ‘inserted’ and ‘noninserted’ state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions. Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high‐throughput approach could allow us to decipher the physiological relevance of protein–protein interactions identified under in vitro conditions or to unveil novel physiological context‐dependent protein–protein interactions that are unknown before.