High-throughput mutational analysis of F₁-ATPase by integrated cell-free protein synthesis and single-molecule...

F₁-ATPase (F₁) is a rotary molecular motor that hydrolyzes adenosine triphosphate (ATP) to drive rotation of the central subunit against the surrounding stator ring. Single-molecule rotation assays of mutated F₁s have elucidated the roles of residues and regions in the chemo-mechanical coupling mechanism, yet comprehensive mutational dissection has been constrained by low-throughput workflows. Here we established a high-throughput platform that integrates cell-free protein synthesis with a multiplexed single-molecule rotation assay. We applied the method to saturation mutagenesis of βE190, the catalytic general base, and βY307, a highly conserved residue at the entrance of a putative phosphate-release tunnel. βE190 substitutions yielded sharply defined outcomes. Only aspartate retained partial activity, highlighting a decisive requirement for negative charge at this catalytic position. By contrast, βY307 variants showed broader tolerance, with activity correlating with side-chain size and hydrophobicity. Moreover, the inactive-state fraction increased for smaller substitutions at βY307, suggesting that the putative tunnel is not catalytically relevant for turnover but may instead be related to occasional phosphate-release events associated with ADP inhibition. Notably, this workflow completes the entire cycle from protein synthesis to functional analysis within 10 h, enabling rapid, comprehensive mutational profiling of rotary ATPases.