Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 24, Pages 9399-9411Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA117.000222
Keywords
pulmonary surfactant; protein-protein interaction; protein complex; lung; membrane structure; fluorescence resonance energy transfer (FRET); fluorescence anisotropy
Categories
Funding
- Spanish Ministry of Economy [BIO2015-67930-R]
- Regional Government of Madrid [S2013/MIT-2807]
- Fundacao para a Ciencia e a tecnologia (Portugal) [FAPESP/20107/2014, SAICTPAC/0019/2015, SFRH/BPD/111301/2015]
- Portuguese Fundacao para a Ciencia e a Tecnologia (FCT) [007630 UID/QUI/00313/2013]
- COMPETE2020-UE
- Portugal 2020/FEDER
- [PTDC/DTP-FTO/2784/2014]
- Fundação para a Ciência e a Tecnologia [PTDC/DTP-FTO/2784/2014, FAPESP/20107/2014] Funding Source: FCT
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Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this work, we report an extensive fluorescence spectroscopy study of both intramolecular and intermolecular SP-B and SP-C interactions, using a combination of quenching and FRET steady-state and time-resolved methodologies. These proteins are compartmentalized in full surfactant membranes but not in pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles, in accordance with their previously described preference for liquid disordered phases. From the observed static self-quenching and homo-FRET of BODIPY-FL labeled SP-B, we conclude that this protein forms homoaggregates at low concentration (lipid:protein ratio, 1:1000). Increases in polarization of BODIPY-FL SP-B and steady-state intensity of WT SP-B were observed upon incorporation of under-stoichiometric amounts of WT SP-C. Conversely, Marina Blue-labeled SP-C is quenched by over-stoichiometric amounts of WT SP-B, whereas under-stoichiometric concentrations of the latter actually increase SP-C emission. Time-resolved hetero-FRET from Marina Blue SP-C to BODIPY-FL SP-B confirm distinct protein aggregation behaviors with varying SP-B concentration. Based on these multiple observations, we propose a model for SP-B/SP-C interactions, where SP-C might induce conformational changes on SP-B complexes, affecting its aggregation state. The conclusions inferred from the present work shed light on the synergic functionality of both proteins in the pulmonary surfactant system.
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