Bilayer-Dependent Recognition of Docosahexaenoic Acid by the Transmembrane Domain of FATP3

Fatty acid Transport Protein 3 (FATP3) is a single-pass transmembrane protein implicated in the uptake and intracellular transport of long-chain fatty acids, yet the molecular contribution of its transmembrane domain (TMD) remains poorly defined. Here, we establish an efficient and reproducible strategy for heterologous expression, purification, and in-vitro reconstitution of FATP3-TMD. FATP3-TMD was over-expressed in Escherichia coli as a TrpLE fusion, liberated by cyanogen-bromide cleavage and polished by one-step reverse-phase HPLC, yielding milligram quantities of highly pure peptide. ¹ H- ¹⁵ N HSQC spectroscopy revealed a well-folded FATP3-TMD in both Fos-choline-14 micelles and DMPC/DHPC bicelles. Strikingly, titration with docosahexaenoic acid (DHA) induced residue-specific chemical-shift perturbations exclusively in bicelles. These data demonstrate that a bilayer-like lipid context is essential for functional recognition of ω-3 fatty acids by the FATP3-TMD and provide a robust platform for mechanistic dissection of FATP3 mediated lipid transport.