Collada Marugán, AinhoaCarrascosa Tejedor, JavierSánchez Puga, PabloLiguori, AlessioGutfreund, PhilippSantamaría, AndreasCastillo Sánchez, José CarlosMaestro, ArmandoCruz Rodríguez, AntonioPérez Gil, Jesús2025-09-222025-09-222025-08Collada A, Carrascosa-Tejedor J, Sánchez-Puga P, Liguori A, Gutfreund P, Santamaría A, Castillo-Sánchez JC, Maestro A, Cruz A, Pérez-Gil J. Escaping from Flatland: the role of proteins SP-B and SP-C in the formation of 3D structures in interfacial pulmonary surfactant films. Journal of Colloid and Interface Science 2026;701:138769. https://doi.org/10.1016/j.jcis.2025.138769.0021-979710.1016/j.jcis.2025.138769https://hdl.handle.net/20.500.14352/124199Deciphering the molecular structure of pulmonary surfactant (PS) at the respiratory air-liquid interface has remained a major challenge since its discovery. This is particularly critical at minimal lung volume and surface area at the end of exhalation, when PS rapidly reorganizes into a 3D membrane network without detaching from the interfacial film, ensuring readiness and stability for subsequent respiratory cycles. Using neutron reflectometry and epifluorescence microscopy in specially designed surface balances, we have investigated the structure of model PS membranes and films at different compression stages, focusing on the key roles of the hydrophobic surfactant proteins SP-B and SP-C in the organization of the system at the interface. The structure of the studied model surfactant films (both analysed orthogonal to the interfacial plane and laterally) strongly depended on composition. We clarified the distinct roles of SP-B and SP-C, revealing that only SP-B, the only protein in surfactant that is indispensable for life, nucleates 3D membrane reservoirs beneath the interface. These findings provide mechanistic insights into how PS maintains interfacial stability during respiration, with potential implications for understanding surfactant dysfunction in respiratory diseases and for designing biomimetic surfactant replacements.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal article1095-7103https://doi.org/10.1016/j.jcis.2025.138769https://www.sciencedirect.com/science/article/pii/S0021979725021605?via%3Dihub#ab0015open access577.112612.2602Lung surfactantHydrophobic proteinsNeutron reflectometryAir-liquid interfaceBreathing dynamicsBioquímica (Biología)BiotecnologíaFisiología2403 Bioquímica2411.17 Fisiología de la Respiración2406 Biofísica