Mapping lipid-dependent operational outcomes of puroindoline-derived tryptophan-rich peptides at model membrane interfaces.

HYPOTHESIS: Membrane-active peptides are often described as "membrane targeting," yet peptide adsorption, vesicle leakage, and colloidal destabilisation are not necessarily coupled. We hypothesised that lipid composition controls access to distinct ensemble-level peptide-LUV interaction outcomes, and that sequence-derived variables bias these outcomes rather than encoding a single universal lytic mechanism.

EXPERIMENTS: Three tryptophan-rich cationic peptides (PuroA, P1, W7) were examined against composition-defined large unilamellar vesicles (LUVs) representing Gram-, Gram+, fungal, red blood cell, and cancer-mimetic membrane classes. Calcein leakage was used as a functional readout of membrane permeabilisation, while zeta potential and dynamic light scattering were used as comparative electrokinetic and intensity-weighted colloidal readouts, respectively. A two-parameter Δζ vs Δsize map was constructed as an operational framework to compare peptide-LUV outcomes across lipid environments.

FINDINGS: Leakage thresholds were strongly membrane-dependent: Gram-/Gram+ surrogates generally reached leakage at lower peptide concentrations than sterol-rich host-like surrogates, whereas fungal membranes showed intermediate behaviour. Zeta potential shifts reported peptide-associated electrokinetic changes but did not monotonically predict leakage, showing that electrostatic engagement alone is insufficient to define permeabilisation outcome. In contrast, large changes in apparent hydrodynamic size were associated with high-leakage outcomes in several bacterial-surrogate systems, consistent with aggregation-associated leakage in LUV suspensions. Substantial leakage also occurred without large size increases, indicating non-aggregation-associated permeabilisation that is consistent with local peptide-induced interfacial perturbation or transient pore-like states, but not definitive of a specific molecular pathway. Overall, the data support a lipid-dependent, operational sequence-interface-function framework for comparing how tryptophan-rich peptides interact with composition-defined membrane models.