Exploring the thermodynamic competition between APP C99 dimerization and membrane partitioning in model liquid-ordered and disordered membranes

Lateral membrane heterogeneity creates distinct lipid environments that modulate both the localization and association of transmembrane proteins. Previous studies have examined how transmembrane proteins partition between ordered and disordered domains, and separate studies have shown that membrane composition can modulate helix association. However, for oligomerizing transmembrane proteins, these two processes are thermodynamically coupled: the phase that most strongly stabilizes the oligomer may also impose a large cost for monomer partitioning. How this competition determines the membrane-domain preference of an associated state remains unclear. Here, we use coarse-grained molecular dynamics simulations along with well-tempered metadynamics and umbrella sampling to quantify this balance for the amyloid precursor protein C-terminal fragment APP C99. We first calculated APP C99 dimerization free-energy landscapes in homogeneous Ld-like DIPC/cholesterol and Lo-like DPPC/cholesterol bilayers with systematically varied cholesterol content. We then calculated the monomer partitioning free energy across a Lo/Ld phase-separated membrane composed of DPPC, DIPC, and cholesterol and combined these quantities through a thermodynamic cycle. APP C99 dimerization is strongly stabilized in Lo-like membranes, with ordered DPPC/cholesterol bilayers providing an additional ∼7−9 kcal/mol stabilization relative to Ld-like bilayers. Despite this stronger ordered-phase dimerization, the free-energy cost of transferring APP C99 monomers into the Lo domain outweighs the stabilizing gain, leaving the dimer thermodynamically favored in the Ld domain. These results establish a thermodynamic framework for resolving the coupling between monomer partitioning and phase-dependent association, providing a roadmap for extending this approach to more diverse membrane compositions and higher-order transmembrane assemblies.