Biointerfacial mechanical modulation of model lipid membranes by gramicidin D: A quartz crystal microbalance with dissipation analysis

Abstract

The rapid evolution of multidrug-resistant bacteria necessitates the development of novel therapeutic strategies. Membrane-active peptides (MAPs) have emerged as promising candidates due to their ability to bypass traditional resistance mechanisms by directly targeting the structural integrity of the bacterial envelope. However, a fundamental challenge in biophysics remains in deciphering how these peptides perturb biological boundaries. The functional outcome of such interactions is dictated by a complex mechanical balance between the peptide and the host lipid architecture. Gramicidin D (GrD), a small and structurally well-defined pentadecapeptide, serves as a prototypical model for MAPs. While traditionally studied through the lens of ion conductivity, its role in large-scale structural remodeling of the membrane remains less understood. To isolate these interactions from complex cellular dynamics, this study utilizes biomimetic membrane models, specifically; Supported Lipid Bilayers (SLBs) and Supported Vesicular Layers (SVLs). These platforms provide the necessary stability for high-resolution, surface-sensitive characterization-most notably overtone-resolved Quartz Crystal Microbalance with Dissipation (QCM-D). A comprehensive, depth-dependent perspective on the membrane's viscoelasticity is gained by probing the lipid architecture at varying distances from the sensor surface through the analysis of multiple overtones. This approach allows for the precise quantification of how peptide partitioning influences the mass, thickness, and mechanical stiffness of the membrane in real-time and non-invasive manner. This study provides a unified mechanistic picture where membrane geometry, surface charge, and peptide density jointly determine the nature of GrD-induced perturbations. The experimental design contrasts planar SLBs with curvature-maintaining SVLs, utilizing both neutral (DOPC) and anionic (DOPC:DOPS 92:08) compositions across two distinct dose-dependent regimes. These biophysical investigations were corroborated by solution-phase experiments. Optical density (OD) measurements indicated a concentration-dependent transition: while low concentrations remained stable, increased peptide levels induced the formation of large macro-aggregates, leading to an initial rise in absorbance followed by a decrease due to sedimentation. The functional integration of GrD was further validated through pyranine dye-leakage assays, which confirmed ion-channel activity under induced pH gradients. Complementary fluorescence microscopy revealed a stark morphological transition from a continuous, homogeneous bilayer to a heterogeneous, disrupted landscape characterized by both large immobile and small mobile aggregates. QCM-D data provided deep insights into the mechanical "fingerprints" of these interactions. In SLB systems, the rigid substrate suppresses large-scale deformations, confining GrD activity to hydration-driven responses within the outer leaflet. In contrast, SVLs accommodate curvature- and elasticity-mediated rearrangements, enabling significantly larger shifts in coupled mass and dissipation that reflect a deeper structural remodeling of the vesicle shell. The introduction of anionic DOPS and Mg2+ ions further modulates this landscape, inducing a charge-dependent regime marked by transient positive frequency excursions. These signatures signify localized hydration rearrangements and an electrostatically constrained insertion mode, where GrD is anchored within a shallow interfacial zone near the headgroups. In this study, the behavior of Gramicidin D (GrD) is shown to be governed by a complex interplay between environmental boundary conditions and peptide polymorphism. GrD-induced membrane perturbation is characterized as a concentration-dependent continuum, ranging from functional channel insertion to large-scale mechanical destabilization. The comprehensive biomechanical framework is established by reconciling acoustic signatures with independent solution-phase assays. Consequently, overtone-resolved QCM-D serves as a sensitive diagnostic platform for distinguishing subtle peptide-insertion kinetics from structural collapse, offering critical insights for the design and evaluation of next-generation membrane-active therapeutic agents.