Manuel Prieto

Pulmonary surfactant (PS) is a lipid–protein complex that forms films reducing surface tension at the alveolar air–liquid interface. Surfactant protein C (SP‐C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N‐terminal segment of SP‐C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C‐terminal region comprises a transmembrane α‐helix that contains a ALLMG motif, supposedly analogous to a well‐studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP‐C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP‐C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self‐quenching and FRET assays to analyze dimerization of native palmitoylated SP‐C and a non‐palmitoylated recombinant version of SP‐C (rSP‐C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP‐C remains primarily monomeric. In contrast, non‐palmitoylated recombinant SP‐C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α‐helical conformation of SP‐C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher‐order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.