[WS4] APPA/PSSJ Joint Workshop Toward International Cooperation in Protein Science

[WS4-02] The High-throughput Genetically Directed Crosslinking Analysis Reveals the Physiological Relevance of the ATP Synthase “Inserted” State

^○Zengyi Chang (State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Center for Protein Science, Peking University)

ATP synthase, a highly conserved protein complex that has a subunit composition of α₃β₃γδεab₂c_8-15 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 F_o sector (ab₂c_8-15). 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 photocrosslinking 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 crosslinked 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 “non-inserted” 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.