Interfacial Charge-Driven Compression-Induced Structural and Morphological Reorganization in Lysozyme–DMPA Mixed Langmuir Films

Protein–lipid interactions play a crucial role in determining the structural and mechanical properties of biological membranes. In this work, mixed Langmuir films composed of lysozyme and the anionic phospholipid 1,2-dimyristoyl- sn -glycero-3-phosphate (DMPA) are fabricated at the air–water (A/W) interface via a cospreading approach to serve as a simplified lipid–protein binary system. The lysozyme-to-DMPA ratio and the subphase pH are systematically varied to modulate the effective interfacial charge and thereby probe its influence on intermolecular interactions and interfacial structure. Surface pressure–area isotherms together with compressibility modulus analysis are used to evaluate phase behavior and mechanical response, while in situ Brewster angle microscopy (BAM) provides insight into the evolution of lateral morphology at the A/W interface. After Langmuir film transfer onto hydrophilic silicon substrates, atomic force microscopy (AFM) and X-ray reflectivity (XRR) are employed to investigate the in-plane morphology and out-of-plane structural organization. The combined results reveal pressure-induced reorganization within the mixed films, characterized by lateral phase separation into domains, together with partial vertical stratification at higher surface pressures, the extent of which depends on the lysozyme-to-DMPA ratio as well as the subphase pH. XRR analysis indicates the formation of a relatively dense mixed protein–lipid base layer accompanied by a less dense overlayer, suggesting redistribution of molecules into an upper region upon compression. Domain observation in AFM images further suggests that the upper layer also contains both protein and lipid components, within which lateral demixing may occur. The reversible hysteresis behavior observed in Π–A isotherm cycles and structural observations confirms that the pressure-induced structural reorganization with compression of the film is reversible in nature. Overall, the findings demonstrate that effective interfacial charge, controlled by protein–lipid composition and subphase pH, plays a crucial role in governing the structural organization and macroscopic behavior of lysozyme–DMPA mixed Langmuir films.