Fig. 5: Molecular dynamics simulations of VLCAD and its proline mutants in the presence of membrane.

a Simulated structure of a wild-type VLCAD monomer in contact with a cardiolipin-containing membrane bilayer (4:1 ratio of phosphatidylcholine [POPC]: tetraoleoyl cardiolipin [TOCL]), as demonstrated by molecular dynamics simulation after 125 ns of equilibration. Residues 446–474 of the α-helical hairpin insert deeply and stably into the lipid membrane. POPC, tan; TOCL, orange; FAD, yellow; substrate, green. b Independently computed electrostatic maps for wild-type VLCAD and lipid membrane, illustrating a complementary potential V between adjacent regions (blue: V > 0; red: V < 0; white, V = 0). c Representative electrostatic interactions between cationic (K, R) residues of VLCAD and cardiolipin headgroups of the lipid membrane or anionic (D, E) amino acids of the protein. To maintain clarity, only the charged phosphates (PO₄²⁻) of TOCL (orange) are shown. d, e Proline mutagenesis disrupts the local structure, resulting in more superficial membrane interaction compared to wild-type VLCAD, as demonstrated by molecular dynamics simulation of the A450P and L462P variants after 125 ns of equilibration. f Comparative geometric quantification of the membrane interactions of wild-type and proline-mutant VLCAD proteins, including, from top to bottom, (1) helical content hlx of the C-terminal region comprised of amino acids 436–488, (2) instantaneous tilt angle θ between a membrane-embedded α-helical segment (residues 460–474) and the membrane surface, (3) height d of the helix center-of-mass with respect to the membrane surface, as defined by a plane through phosphate atoms in the proximal membrane leaflet, whereby values with d < 0 lie below the lipid headgroups, and (4) solvent-accessible surface area A_SASA buried at the VLCAD-lipid interface. Trajectories were smoothed by averaging over 0.5 ns bins, and bands reflect the standard deviation within these bins. Calculations were facilitated by the collective variables module of VMD⁷¹ where applicable⁷². Adjacent bar graphs depict mean values ± s.d. of the geometric parameters, calculated over the last 80 ns equilibration trajectory (n = 320).