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. ¹H-¹⁵N 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.