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

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

Yang Liu

2020

Scholarcy highlights

  • 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 and a membrane-integrated Fo sector
  • 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
  • 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. 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
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