Morán Lalangui, Juranny MichelleCoutinho, AnaPrieto, ManuelFedorov, AlexanderPérez Gil, JesúsLoura, Luís M. S.García Álvarez, María Begoña2024-07-012024-07-012023-12-20Morán-Lalangui M, Coutinho A, Prieto M, Fedorov A, Pérez-Gil J, Loura LMS, et al. Exploring protein–protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching. Protein Science. 2024; 33(1):e4835.0961-836810.1002/pro.4835https://hdl.handle.net/20.500.14352/105402Acknowledgments: FPI fellowship from the Spanish Ministry of Science and Innovation. COMPETE2020-Operational Program for Competitiveness and Internationalization, funding by the European Regional Development Fund. This work has been supported by a working visit bursary from EBSA.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.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal article1469-896Xhttps://doi.org/10.1002/pro.4835https://onlinelibrary.wiley.com/doi/full/10.1002/pro.4835open access577.112577.2FRETOligomerization stateProtein–protein interactionPulmonary surfactantSelf-quenchingSurfactant protein C (SP-C)Bioquímica (Química)Biología molecular (Biología)2302 Bioquímica2415 Biología Molecular