Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers

Pulmonary surfactant-associated protein B (SP-B) has been incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) by two different procedures to characterize the dependence of lipid–protein interactions on the method of reconstitution. In method A the protein was dissolved in a small volume of either methanol or 60% (v/v) acetonitrile and injected into an aqueous phase containing phospholipid vesicles. In method B the vesicles were prepared by injection of a mixture of phospholipid and SP-B dissolved in methanol or aqueous acetonitrile. Both methods of reconstitution led to the extensive interaction of SP-B with PC bilayers as demonstrated by co-migration during centrifugation, marked protection against proteolysis, change in the fluorescence emission intensity of SP-B, and protection of SP-B tryptophan fluorescence from quenching by acrylamide. SP-B promoted the rapid adsorption of DPPC on an air/liquid interface irrespective of the method of protein reconstitution. However, the interfacial adsorption activity of SP-B reconstituted by method B remained stable for hours, but that of SP-B prepared by method A decreased with time. Electron microscopy showed that the injection of SP-B into an aqueous phase containing PC or DPPC vesicles (method A) induced a rapid aggregation of vesicles. By contrast, a much longer time was required for detecting vesicle aggregation when the protein was reconstituted by co-injection of SP-B and phospholipids (method B). The presence of 5% (w/w) SP-B in DPPC bilayers prepared by method B broadened the differential scanning calorimetry thermogram and decreased the enthalpy of the transition. In contrast, the injection of SP-B into preformed DPPC vesicles (method A) did not influence the gel-to-liquid phase transition of DPPC bilayers. Taken together, these results indicate that the mode and extent of interaction of SP-B with surfactant phospholipids depends on the conditions of preparation of lipid/protein samples, and that care should be taken in the interpretation of findings from reconstituted systems on the role of these surfactant proteins in the alveolar space.

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Effect of Cholesterol and Palmitoylation on the Structure, Orientation and Lipid-Protein Interactions of Pulmonary Surfactant Protein SP-C

Biophysical Journal ◽

10.1016/j.bpj.2012.11.388 ◽

2013 ◽

Vol 104 (2) ◽

pp. 63a-64a

Author(s):

Nuria Roldán López ◽

Elena López Rodríguez ◽

Jesús Pérez-Gil ◽

Begoña Garcia-Alvarez

Keyword(s):

Protein Interactions ◽

Pulmonary Surfactant ◽

Surfactant Protein ◽

Lipid Protein Interactions ◽

Structure Orientation ◽

Pulmonary Surfactant Protein

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Synthesis of Nanoceramic Particles by Intravesicular Precipitation

MRS Proceedings ◽

10.1557/proc-180-637 ◽

1990 ◽

Vol 180 ◽

Cited By ~ 1

Author(s):

Suhas Bhandarkar ◽

Iskandar Yaacob ◽

Arijit Bose

Keyword(s):

Aqueous Phase ◽

Magnetic Particles ◽

Egg Yolk ◽

Ferric Nitrate ◽

Close Match ◽

Egg Yolk Phosphatidylcholine ◽

Starting Solution ◽

Single Compartment ◽

Multiple Particles ◽

Direct Formation

ABSTRACTNanometer sized magnetic particles have been fabricated by aqueous phase coprecipitation inside single compartment vesicles. These vesicles were generated by sonicating egg yolk phosphatidylcholine molecules in an appropriate ionic solution containing the reactant cations. Using cobalt and ferric nitrate as the starting solution, the reaction product was the desired cobalt ferrite; direct formation of the oxide is potentially a very important step. For the barium and ferric nitrate systems, the reaction product could not be identified easily. However, we have not been able to produce barium ferrite directly. In an attempt to compare the effect of drastic changes in the microenvironment, we have also completed aqueous phase precipitation in multilamellar vesicles. For the aluminum nitrate system, precipitation takes place only in the outermost “annular” space. Multiple particles are formed, and the resultant diffraction pattern shows a close match with an aluminum hydroxide chloride complex. This differs significantly from the product in single compartment vesicles, where β-Al2O3 was formed, and in free precipitation which resulted in Al(OH)3.

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Synthesis of spin-labelled 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine

Canadian Journal of Biochemistry ◽

10.1139/o80-020 ◽

1980 ◽

Vol 58 (2) ◽

pp. 143-146 ◽

Cited By ~ 6

Author(s):

D. J. Vaughan ◽

N. Z. Stanacev

Keyword(s):

Protein Interactions ◽

Egg Yolk ◽

Molar Ratio ◽

Resonance Spectroscopy ◽

Scanning Calorimetry ◽

Spin Resonance ◽

Dimyristoyl Phosphatidylcholine ◽

Label Probe ◽

Lipid Protein Interactions ◽

Good Agreement

Chemical synthesis of spin-labelled lecithin 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)-stearoyl)-phosphatidylcholine was achieved by reaction of equimolar amounts of purified egg yolk lyso-lecithin with commercially available 16-(N-oxyl-4′,4′-dimethyloxazolidine)stearic acid activated with N,N-carbonyldiimidazole. After purification by silicic acid column chromatography, spin-labelled lecithin was obtained in 52.2% yield and had an ester:phosphorus:spin molar ratio of 2.0:1.1:1.0. When dimyristoyl-phosphatidylcholine and dimyristoyl-phosphatidylcholine:cholesterol (10:1) liposomes, containing 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine as a probe, were examined for thermotropic changes monitored by electron spin resonance spectroscopy, transition temperatures of 24.0 and 24.2 °C, respectively, were obtained in a very good agreement with previously reported values obtained with different probes and by different techniques, and with our own differential scanning calorimetry measurements. The potential usefulness of synthetic 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine as a spin-label probe in studies of lipid–protein interactions in biological membranes was discussed.

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Tryptophan fluorescence study on the interaction of pulmonary surfactant protein A with phospholipid vesicles

Biochemical Journal ◽

10.1042/bj2960585 ◽

1993 ◽

Vol 296 (3) ◽

pp. 585-593 ◽

Cited By ~ 54

Author(s):

C Casals ◽

E Miguel ◽

J Perez-Gil

Keyword(s):

Protein A ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Surfactant Protein ◽

Intrinsic Fluorescence ◽

Surfactant Protein A ◽

Phospholipid Vesicles ◽

Fluorescence Emission Spectrum ◽

Fluorescence Measurements ◽

Fluorescence Characteristics

The fluorescence characteristics of surfactant protein A (SP-A) from porcine and human bronchoalveolar lavage were determined in the presence and absence of lipids. After excitation at either 275 or 295 nm, the fluorescence emission spectrum of both proteins was characterized by two maxima at about 326 and 337 nm, indicating heterogeneity in the emission of the two tryptophan residues of SP-A, and also revealing a partially buried character for these fluorophores. Interaction of both human and porcine SP-A with various phospholipid vesicles resulted in an increase in the fluorescence emission of tryptophan without any shift in the emission wavelength maxima. This change in intrinsic fluorescence was found to be more pronounced in the presence of dipalmitoyl phosphatidylcholine (DPPC) than with dipalmitoyl phosphatidylglycerol (DPPG), DPPC/DPPG (7:3, w/w) and 1-palmitoyl-sn-glycerol-3-phosphocholine (LPC). Intrinsic fluorescence of SP-A was almost completely unaffected in the presence of egg phosphatidylcholine (egg-PC). In addition, we demonstrated a shielding of the tryptophan fluorescence from quenching by acrylamide on interaction of porcine SP-A with DPPC, DPPG or LPC. This shielding was most pronounced in the presence of DPPC. In the case of human SP-A, shielding was only observed on interaction with DPPC. From the intrinsic fluorescence measurements as well as from the quenching experiments, we concluded that the interaction of some phospholipid vesicles with SP-A produces a conformational change on the protein molecule and that the interaction of SP-A with DPPC is stronger than with other phospholipids. This interaction appeared to be independent of Ca2+ ions. Physiological ionic strength was found to be required for the interaction of SP-A with negatively charged vesicles of either DPPG or DPPC/DPPG (7:3, w/w). Intrinsic fluorescence of SP-A was sensitive to the physical state of the DPPC vesicles. The increase in intrinsic fluorescence of SP-A in the presence of DPPC vesicles was much stronger when the vesicles were in the gel state than when they were in the liquid-crystalline state. The effect produced by SP-A on the lipid vesicles was also dependent on temperature. The aggregation of DPPC, DPPC/DPPG (7:3, w/w) or dimyristoyl phosphatidylglycerol (DMPG) was many times higher below the phase-transition temperature of the corresponding phospholipids. These results strongly indicate that the interaction of SP-A with phospholipid vesicles requires the lipids to be in the gel phase.

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