Spectral Phasor Analysis Reveals Deviation from Ideal Two-State Membrane Ordering Induced by Polybia-MP1 and Analogues.

Laurdan generalized polarization (GP) is widely used to assess lipid packing and membrane order in model membranes. However, because conventional GP analysis relies on intensities at two selected wavelengths, it may overlook spectral changes not adequately captured by blue- and red-shifted emission contributions. Spectral phasor analysis, which maps the entire emission spectrum onto a model-free (G, S) plane, offers a complementary and potentially more sensitive readout of membrane order. Here, we applied spectral phasor analysis to Laurdan-labeled large unilamellar vesicles (LUVs) composed of E. coli polar lipid extract, in the absence and presence of the wasp-derived antimicrobial peptide Polybia-MP1 and four related analogues in which individual lysine residues were replaced by histidine. This design allowed us to investigate how the position of histidine influences peptide-induced membrane perturbations while maintaining the same net charge. Phasor coordinates were obtained across a temperature series (20, 37, 60 °C) and, independently, across a lipid-titration series. For each dataset, we fitted the phasor trajectory of the peptide-free LUV reference and decomposed every peptide-containing phasor point into two orthogonal components relative to this trajectory: a component along the line, equivalent to the classical lever rule and quantifying the ordered/disordered fraction, and a perpendicular component quantifying deviation from ideal two-state mixing. The peptide-free LUV trajectory was essentially linear across temperature (fit residuals ~10⁻⁴), consistent with simple two-state thermal behavior. Four of the five peptides remained close to this trajectory, indicating that they modulate the same ordered/disordered equilibrium already present in the peptide-free membrane. MP1, in contrast, showed a perpendicular deviation five- to fifty-fold larger than any other peptide-temperature combination, most pronounced at 60 °C, despite a comparatively modest change in classical GP alone — a distinction invisible to the two-point ratiometric metric. Titration-series phasor data are currently being processed under the same lever-rule/deviation framework, and experiments are underway to define a third reference state, to test whether MP1's deviation reflects a genuine three-component mixing regime describable by a tie-triangle. In this work, we applied an explicit orthogonal deviation metric to Laurdan spectral phasors across lipid titrations, complementing recent ternary-phasor approaches developed for classifying intrinsic lipid mesophases.