Bilayer Elastic Properties Modulate Interface Energetics of Transmembrane β-Barrels

Assembly energetics of transmembrane proteins occurs through the orchestrated molecular interplay of the protein's membrane partitioning free energy and the mechanical and physicochemical properties of the bilayer. While amphipathic residues help accommodate diametrically opposite forces and the steep energy gradient at the membrane-water interface, the magnitude to which the intrinsic curvature and elastic energy of the bilayer regulates protein thermodynamics at the interface remains poorly understood. Here, using PagP as a model β-barrel, we compare the per-residue energetics of an interface site across membranes with(out) distinct curvature stress, lipid packing, and headgroup chemistry. In vesicles, the measured interface energetics correlates well with known free energy scales. Interestingly, unlike vesicles, we show that planar bicelles impose fundamentally distinct membrane-protein coupling regimes: (i) PagP experiences altered folding with a stabilized membrane-associated intermediate and (ii) side chain transfer free energies are restructured in a residue-specific manner. Additionally, the energetics are unaffected by the presence of nonlamellar lipids. Our results reveal an unexpected dependence of partitioning energetics on both bilayer elastic energy and lipid packing defects, demonstrating how subtle changes in membrane physical properties could redirect protein folding pathways and thermodynamics. Our findings underscore the need for caution when extrapolating side chain transfer free energies from simplified model membranes to crowded, compositionally complex in vivo environments.