Characterization of the In SituStructural and Interfacial Properties of the Cationic Hydrophobic Heteropolypeptide, KL4, in Lung Surfactant Bilayer and Monolayer Models at the Air−Water Interface: Implications for Pulmonary Surfactant Delivery

This study examines the various equilibrium in situsecondary structures of the pharmaceutical heteropolypeptide, KL 4, in the solid state, in solution, and in the monolayer state alone and mixed with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG). In situsurface circular dichroism spectroscopy, using a method first reported by Damodaran (Damodaran, S. Anal. Bioanal. Chem.2003, 376, 182−188), of equilibrated KL 4, DPPC/KL 4, POPG/KL 4, and DPPC/POPG/KL 4monolayers at the air−water interface was used to examine the in situtwo-dimensional conformation of KL 4. Gravimetric vapor sorption by solid KL 4was used to analyze the effects of water molecules on the conformation of KL 4when confined as a monolayer at the surface of water. Solid-state KL 4conformation was determined by X-ray powder diffraction (XRPD). The equilibrium interfacial and spreading properties were measured at 25 °C, 37 °C, and 45 °C using the Wilhelmy plate method and Langmuir film balance. Equilibrium phase transition temperatures were measured using differential scanning calorimetry (DSC). It was found that solid-state KL 4, which takes up very little water, exhibits β-sheet and α-helix secondary structures, whereas KL 4in solution appears to exist only as an α-helix. KL 4forms a stable, insoluble monolayer, exhibiting β-sheet and aperiodic structures. These structures provide KL 4, when confined in two-dimensions, the structural flexibility to maximize favorable cationic lysine−water interactions and favorable leucine−leucine hydrophobic and van der Waals interactions; while effectively “shielding” the leucine residues away from water. In DPPC/KL 4monolayers, KL 4retains its native β-sheet and aperiodic structures, consistent with phase separation of DPPC and KL 4in bilayers and monolayers. In POPG/KL 4monolayers, KL 4exhibits an increase in aperiodic secondary structures (loss of β-sheet) to maximize favorable electrostatic interactions, consistent with the observed negative deviations from ideal monolayer mixing.