High‐throughput mutational analysis of F ₁ ‐ ATPase by integrated cell‐free protein synthesis and single‐molecule rotation assay

F 1 ‐ATPase (F 1 ) 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 F 1 s 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.