Molecular Insights into the Bacterial Membrane Interactions of Proline-Stabilized Graphene: Effects of Proline Oligomerization

Abstract Multidrug-resistant bacterial infections pose a major global health challenge. Graphene nanosheets exhibit promising antibacterial activity through membrane disruption, but their practical application is limited by poor aqueous stability and biocompatibility. A recent study [Nature 2025, 645 (8082), 915–921] has suggested that amino acids such as proline can enhance nanomaterial stability and biocompatibility through noncovalent interactions; however, their influence on graphene–membrane interactions remains unclear. Here, all-atom molecular dynamics simulations were performed to investigate the effects of proline oligomerization on the insertion of graphene nanosheets into a bacterial membrane. Monomeric (2 M), dimeric (1 M), and trimeric (0.67 M) proline systems containing equivalent concentrations of proline monomer units were examined. Graphene successfully inserted into the membrane in monomeric and dimeric proline solutions, inducing membrane deformation, altered lipid ordering, and enhanced lipid diffusion. Membrane insertion followed four stages: free movement, slow insertion, rapid insertion, and stable insertion. The average insertion time increased from 12.7 ± 3.1 ns without proline to 56.3 ± 22.5 ns and 169.7 ± 55.0 ns in monomeric and dimeric proline systems, respectively. In contrast, graphene failed to penetrate the membrane in the trimeric proline system within the simulation time scale. Energetic analyses revealed that membrane insertion is governed by competition between graphene–solution and graphene–membrane interactions. Increasing proline oligomerization strengthened graphene–proline interactions and reduced the energetic advantage of membrane insertion, thereby weakening the thermodynamic driving force for membrane penetration. Proline molecules remained in the aqueous phase and detached from the graphene surface during insertion, indicating a stabilizing rather than a direct membrane-penetrating role. These findings demonstrate that graphene can retain its membrane-disruptive activity while benefiting from proline-induced stabilization, provided that proline oligomerization remains limited. This work provides the first evidence of molecular-level insights into the design of amino acid-non-covalently-stabilized graphene-based nanomaterials for potentially disrupting bacterial membranes.