ArticlebioRxiv : the preprint server for biology2025
High-throughput discovery of transmembrane helix dimers from human single-pass membrane proteins with TOXGREEN sort-seq.
Article in bioRxiv : the preprint server for biology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
The oligomerization of the transmembrane helices of single-pass membrane proteins is crucial to biological function and its misregulation can lead to many diseases. The study of transmembrane helix oligomerization is facilitated by the availability of genetic reporter assays, which are essential tools for understanding the organization and biology of single-pass systems. In particular, reporter assays are crucial for mapping the oligomerization interfaces of transmembrane helices through scanning mutagenesis but their application is limited by the need to clone and measure each construct individually. Here, we present "TOXGREEN sort-seq", a high-throughput version of the TOXGREEN assay that enables the direct measurement of transmembrane helix oligomerization in large libraries using fluorescence-activated cell sorting and next-generation sequencing. We show that TOXGREEN sort-seq is robust and reproduce the direct measurements of individual constructs with good accuracy and sensitivity. The method produced high-quality mutational profiles from a library of 17,400 constructs designed to probe the interface of 100 potential GAS
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