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oai:docta.ucm.es:20.500.14352/1054022024-07-01T23:53:15Zcom_20.500.14352_14col_20.500.14352_15

Exploring protein–protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching Morán Lalangui, Juranny Michelle Coutinho, Ana Prieto, Manuel Fedorov, Alexander Pérez Gil, Jesús Loura, Luís M. S. García Álvarez, María Begoña Acknowledgments: 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. 2024-07-01T15:30:25Z 2024-07-01T15:30:25Z 2023-12-20 journal article Morán-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. 0961-8368 10.1002/pro.4835 https://hdl.handle.net/20.500.14352/105402 1469-896X https://doi.org/10.1002/pro.4835 https://onlinelibrary.wiley.com/doi/full/10.1002/pro.4835 eng info:eu-repo/grantAgreement/MICINN//PID2021-124932OB-100 P2018/NMT-4389 info:eu-repo/grantAgreement/FCT//UIDB/00313%2F2020 info:eu-repo/grantAgreement/FCT//UIDB/04565%2F2020 info:eu-repo/grantAgreement/FCT//UIDP/00313%2F2020 info:eu-repo/grantAgreement/FCT//UIDP/04565%2F2020 info:eu-repo/grantAgreement/LA//P/0140%2F2020 http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 International Wiley