Morán Lalangui, Juranny MichelleCoutinho, AnaPrieto, ManuelFedorov, AlexanderPérez Gil, JesúsLoura, Luis M. S.García Álvarez, María Begoña2025-05-302025-05-302023-11-20Morán-Lalangui, M., Coutinho, A., Prieto, M., Fedorov, A., Pérez-Gil, J., Loura, L. M. S., & García-Álvarez, B. (2024). Exploring protein–protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching. Protein Science, 33(1). https://doi.org/10.1002/PRO.48350961-836810.1002/pro.4835https://hdl.handle.net/20.500.14352/120659Begoña García-Álvarez and Jesús Pérez-Gil thanks Spanish Ministry of Science and Innovation Grant (PID2021-124932OB-100) and Community of Madrid Grant (Nanobiocargo P2018/NMT-4389) for funding this study. Mishelle Morán-Lalangui is a recipient of a FPI fellowship from the Spanish Ministry of Science and Innovation. Luís M. S. Loura acknowledges funding by the European Regional Development Fund, through COMPETE2020-Operational Program for Competitiveness and Internationalization, and Portuguese funds via FCT-Fundação para a Ciência e a Tecnologia, under projects UIDB/00313/2020 and UIDP/00313/2020 (Portugal). Ana Coutinho and Manuel Prieto acknowledge funding from FCT-Fundação para a Ciência e a Tecnologia, under projects UIDB/04565/2020, UIDP/04565/2020 and LA/P/0140/2020 (Portugal). 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/10.1002/pro.4835open access616.24612.793577FRETOligomerization stateProtein–protein interactionPulmonary surfactantSelfquenchingSurfactant protein C (SP-C)NeumologíaBioquímica (Biología)Biología molecular (Biología)3205.08 Enfermedades Pulmonares2403 Bioquímica2415 Biología Molecular2302.27 Proteínas