Biointerfacial mechanical responses of supported lipid membranes to Gramicidin D revealed by QCM-D

Gramicidin D (GrD) is a well-established model ion channel-forming peptide whose mechanical interactions with supported lipid membrane architectures remain less well characterized. Here, we combine overtone-resolved Quartz Crystal Microbalance with Dissipation (QCM-D), fluorescence microscopy, and solution-phase assays to examine how GrD perturbs supported lipid bilayers (SLBs) and supported vesicular layers (SVLs) formed from neutral DOPC and anionic DOPC:DOPS (92:08). On neutral SLBs at low peptide levels, GrD induces mild outer-leaflet softening consistent with β^6.3 channel formation, producing subtle acoustic signatures that evolve with slow kinetics. In contrast, neutral SVLs exhibit immediate hydration and thickening of the vesicle shell, reflected by large dissipation increases and overtone-dependent frequency shifts. Increasing peptide levels drive both platforms into a mechanically disruptive regime characterized by distinct overtone divergence and the formation of highly dissipative peptide-lipid assemblies, including rapid disruptive remodeling on SLBs and collapse of peptide-rich outer shells on SVLs. Membrane charge further modulates these responses. Anionic SLBs and SVLs confine GrD to shallow, leaflet-restricted perturbations near the headgroup region, yielding smaller overall mass changes but strong, overtone-specific viscoelastic heterogeneity. Solution-phase turbidity and proton permeability assay are consistent with functional channel activity dominating at lower peptide levels, whereas higher peptide levels align with non-conducting, mechanically disruptive interaction states. Together, these results show that GrD exhibits distinct, architecture-dependent mechanical interaction modes governed by peptide density, membrane geometry, and surface charge, and demonstrate how QCM-D measurements on complementary SLB and SVL platforms resolve nanoscale hydration and mechanical response modes that remain inaccessible to conventional ensemble assays.