Characterization of the in situ structural 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 situ secondary structures of the pharmaceutical heteropolypeptide, KL4, in the solid state, in solution, and in the monolayer state alone and mixed with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG). In situ surface circular dichroism spectroscopy, using a method first reported by Damodaran (Damodaran, S. Anal, Bioanal. Chem. 2003, 376, 182-188), of equilibrated KL4, DPPC/KL4, POPG/KL4, and DPPC/POPG/KL4 monolayers at the air-water interface was used to examine the in situ two-dimensional conformation of KL4. Gravimetric vapor sorption by solid KL4 was used to analyze the effects of water molecules on the conformation of KL4 when confined as a monolayer at the surface of water. Solid-state KL4 conformation was determined by X-ray powder diffraction (XRPD). The equilibrium interfacial and spreading properties were measured at 25 degrees C, 37 degrees C, and 45 degrees 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 KL4, which takes up very little water, exhibits beta-sheet and alpha-helix secondary structures, whereas KL4 in solution appears to exist only as an alpha-helix. KL4 forms a stable, insoluble monolayer, exhibiting beta-sheet and aperiodic structures. These structures provide KL4, 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/KL4 monolayers, KL4 retains its native,beta-sheet and aperiodic structures, consistent with phase separation of DPPC and KL4 in bilayers and monolayers. In POPG/KL4 monolayers, KL4 exhibits an increase in aperiodic secondary structures (loss of beta-sheet) to maximize favorable electrostatic interactions, consistent with the observed negative deviations from ideal monolayer mixing.