Abstract
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.
Highlights
We addressed the possibility of quenching of Marina Blue SP-C fluorescence by WT SP-B
Previous studies had confirmed that the interfacial activities of the pulmonary surfactant complexes are optimal when both proteins SP-B and SP-C are present in their physiological ratio [20]
The possibility that SP-B and SP-C could cooperate through formation of possible SP-B1⁄7SP-C complexes in lipid membranes has been explored in the present work by quenching and FRET analysis
Summary
The fluorescence anisotropy of both labeled proteins could be adequately described by two rotational correlation times: a faster component, 1 Ͻ 1 ns, and a slower one, 2 Х 6 –9 ns (Fig. 2). This heterogeneity of both fluorescence emission and rotational depolarization kinetics of the labeled proteins points to an average interfacial location of both fluorophores, characterized by steep variations in polarity and microviscosity. The decay kinetics of BODIPY-FL SP-B fluorescence becomes faster upon increasing the concentration of labeled protein, and the variation of the decay parameters was analyzed with a Stern-Volmer equation for dynamical (collisional) quenching,. Combination of this equilibrium with Equation 5
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
