Fluorescence readouts of KL4-induced changes in lipid order and fluidity in surfactant-like bilayers relevant to ARDS and COVID-19 lung injury.

KL4, a 21-residue mimetic of the C-terminus of surfactant protein B, is effective in treating infant respiratory distress syndrome (RDS). It works by reducing alveolar surface tension and improving oxygen exchange. Here, we use steady-state fluorescence measurements to probe how KL4 modulates bilayer order and organization in pulmonary surfactant-like lipid mixtures with varying acyl chain saturation. We show how fluorescence-based methods can distinguish between a more surface-associated and a more deeply inserted mode of interaction of KL4 in phospholipid membranes with varying acyl chain saturation. Specifically, in saturated liposomes with a tail-labeled pyrene analog, KL4 addition decreases excimer-to-monomer (Ie/Im) ratios and produces larger changes in the steady-state anisotropy of rhodamine-labeled lipids, consistent with reduced lipid mobility and peptide-induced changes in bilayer packing, and is qualitatively consistent with previous ssNMR/EPR studies of KL4. Conversely, fluorescence results for unsaturated liposomes show smaller changes in Ie/Im and anisotropy, suggesting greater interfacial interaction between KL4 and a more modest impact on bilayer order. Increasing concentrations of KL4 in unsaturated lipids result in a notable decrease in rhodamine headgroup emission intensity, suggesting local environmental shifts or collisional quenching, consistent with a more surface-proximal peptide. Overall, these fluorescence readouts are sensitive to KL4-induced changes in bilayer order, lipid dynamics, and lateral organization, but do not, by themselves, define peptide orientation or insertion depth. These assays require minimal sample and simple equipment, making them practical for monitoring how KL4 and similar surfactant mimics influence bilayer behavior across different lipid compositions and for screening lipid and peptide combinations in future models of healthy and diseased surfactant relevant to neonatal RDS and adult ARDS, including COVID-19.