Chain Length, Unsaturation, and Conformational State Modulate ω-3 FFA Effects on POPC Bilayers

Abstract Free fatty acids (FFAs), specifically the ω-3 forms α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are important modulators of lipid membrane structure and dynamics, yet how these three species compare within a single, consistent framework remains unclear. Here, we employed atomistic molecular dynamics simulations to investigate the effects of ALA, EPA, and DHA (5–20 mol %) on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers in single-FFA and mixed DHA/EPA systems. FFA incorporation induces concentration-dependent bilayer condensation, marked by decreased area and volume per lipid, increased thickness, and enhanced acyl chain order, with ALA producing the strongest effects. All three FFAs promote reorientation of POPC headgroups toward embedded FFA carboxyl groups. Using a conformational classification scheme (stretched, intermediate, coiled), we show that local ordering of neighboring POPC chains and the FFAs’ own ordering strongly depend on conformational state. FFAs exhibit higher lateral mobility than POPC. Passive transbilayer diffusion depends on FFA type and concentration; only ALA is nonmonotonic. In mixed DHA/EPA systems, lateral diffusion of EPA is enhanced relative to single-FFA systems, whereas passive diffusion of DHA is reduced. These results show that chain length and unsaturation mainly modulate FFA effects on bilayer structure and dynamics, whereas conformational state additionally shapes local acyl-chain ordering, relevant to membrane behavior under elevated and pathological FFA levels.