Self-aggregation of a recombinant form of the propeptide NH2-terminal of the precursor of pulmonary surfactant protein SP-B: a conformational study (original) (raw)
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A model for the structure and mechanism of action of pulmonary surfactant protein B
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2015
Surfactant protein B (SP-B), from the saposin-like family of proteins, is essential to facilitate the formation and proper performance of surface active films at the air-liquid interface of mammalian lungs, and lack of or deficiency in this protein is associated with lethal respiratory failure. Despite its importance, neither a structural model nor a molecular mechanism of SP-B is available. The purpose of the present work was to purify and characterize native SP-B supramolecular assemblies to provide a model supporting structure-function features described for SP-B. Purification of porcine SP-B using detergent-solubilized surfactant reveals the presence of 10 nm ring-shaped particles. These rings, observed by atomic force and electron microscopy, would be assembled by oligomerization of SP-B as a multimer of dimers forming a hydrophobically coated ring at the surface of phospholipid membranes or monolayers. Docking of rings from neighboring membranes would lead to formation of SP-B...
The Journal of biological chemistry, 2018
Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its non-lipidic 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 while 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 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 th...
Biochemistry, 2005
Pulmonary surfactant protein SP-B is absolutely required for proper function of surfactant in the alveoli, and is an important component of therapeutical surfactant preparations used to treat respiratory pathologies. To explore inherent structural and functional determinants within the amino acid sequence of mature SP-B, porcine SP-B has been subjected to extensive disulfide reduction under highly denaturing conditions and to cysteine carboxyamidomethylation, and the structure, lipid-protein interactions, and surface activity of this modified form have been characterized. Refolding of the reduced protein yielded a form (SP-Br) with secondary structure practically identical to that of the native disulfide-linked SP-B dimer. Reduced SP-Br exhibited higher structural flexibility than native SP-B, as indicated by a higher susceptibility of fluorescence emission to quenching by acrylamide and biphasic behavior during interaction of the protein with lipid bilayers and monolayers. SP-Br had, however, effects similar to those of native SP-B on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) bilayers. SP-Br was more effective than native SP-B in promoting interfacial adsorption of phospholipid bilayers into interfacial films, presumably because of its higher structural flexibility, and retained the ability of native SP-B to stabilize DPPC interfacial films compressed to pressures near collapse against spontaneous relaxation. SP-Br also mimicked the behavior of native SP-B in lipid-protein films subjected to dynamic compressionexpansion cycling in a captive bubble surfactometer, but only in the presence of phosphatidylglycerol (PG), the main anionic phospholipid in surfactant. The presence of PG appears to be required for SP-Br to acquire the appropriate tertiary folding to produce progressively more efficient lipid-protein films capable of reaching very high pressures upon limited compression with almost no hysteresis.
Interactions of Pulmonary Surfactant Protein A with Phospholipid Monolayers Change with pH
Biophysical Journal, 1999
The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the -A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the -A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.
Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1995
The structure of hydrophobic pulmonary surfactant-associated proteins SP-13 and SP-C have been studied in different acetonitrile (ACN)/water and trifluorethanol (TFE)/water mixtures by circular dichroism and fluorescence spectroscopy to analyze the conformational flexibility of these proteins in response to changes in solvent composition. SP-B presented a very stable conformation in all the assayed ACN/water mixtures and in TFE/water mixtures containing until 70% TFE, showing around 40% α-helix. When SP-B was transferred to mixtures containing more than 70% TFE, the percent of α-helix in SP-B increased up to 60%. The fluorescence emission spectra of SP-B in the different solvents showed that tryptophan residues are more sensitive to solvent changes than those of tyrosine, reflecting differential effects on different protein microenvironments. The effect of solvent changes on the two tryptophan populations detected by fluorescence spectra was also different. A model for the folding of SP-B dimers, dominated by intra- and intermolecular disulphide bonds, is proposed. Surfactant protein SP-C revealed a secondary structure much more sensitive to solvent composition than SP-B. It had a main a-helical conformation in ACN/water solvents which was up to 63% in mixtures containing more than 60% ACN. When the protein was transferred to solvents containing less than 60% ACN, its secondary structure possessed less percent of α-helix and an increased percent of β-structure. On the other hand, SP-C had a main /3-sheet secondary structure in all the assayed TFE/water mixtures, with 30–40% α-helix and around 50% β-structure. The strong dependence of SP-C conformation on the nature of the solvent is interpreted to arise from its high hydrophobicity and the possible occurrence of protein-protein interactions.
1 BIOPHYSICAL CHARACTERIZATION OF PEPTIDE MIMICS OF LUNG SURFACTANT PROTEIN-B By
2008
I thank my mentor Dr. Joanna Long for providing me the opportunity for working in her laboratory. I thank her for her support, patience, sense of humor, and kindness. I hope to acquire some of those qualities that she has as I continue to mature and develop. I would also like to offer my gratitude to members of my committee: Dr. Arthur Edison, Dr. Robert McKenna, Dr. Susan Frost and Dr. Ron Castellano. I appreciate their support and their help. I am particularly grateful for Dr. Susan Frost for giving me some extra support with regards to my presentation and for her willingness to afford some of her time to look over my dissertation. I want to extend my heartfelt gratitude and sincere thanks to members of the laboratory
Influence of Pulmonary Surfactant Protein B on Model Lung Surfactant Monolayers
Langmuir, 2002
Pressure-area isotherms, Brewster angle microscopy, and grazing incidence X-ray diffraction measurements reveal that human lung surfactant protein SP-B1-78 and the dimer of the amino terminus dSP-B1-25 modify the phase behavior of lipid mixtures consisting of dipalmitoylphosphatidylcholine/palmitoyl-oleylphosphatidylglycerol/palmitic acid (DPPC/POPG/PA). The addition of SP-B increases the fraction of fluid phase in the liquid-expanded/liquid-condensed two-phase region. Brewster angle microscopy enabled the visualization of a fluid network, which separates the condensed phase domains. This network is stabilized by SP-B adsorption. GIXD measurements show that SP-B also alters the structure of the condensed chain lattice leading to higher tilt and increased area per hydrocarbon chain. The comparison of SP-B1-78 with the shorter peptide dSP-B1-25 exhibits, that the dimer alters the lipid order more drastically. The larger effects found for dSP-B1-25 were explained using a model that assumes a partial incorporation of the peptide into the layer. The specific behavior of the dimer could enhance the activity of the peptide as found in recent animal model studies. This is the first investigation showing a systematic influence of SP-B on the condensed chain lattice of phospholipids, thus verifying that SP-B not only interacts with the expanded phase, but also interactions with the condensed phase lipids have to be taken into account which might be essential for proper peptide function.
Biophysical Journal, 2003
The relationship among protein oligomerization, secondary structure at the interface, and the interfacial behavior was investigated for spread layers of native pulmonary surfactant associated proteins B and C. SP-B and SP-C were isolated either from butanol or chloroform/methanol lipid extracts that were obtained from sheep lung washings. The proteins were separated from other components by gel exclusion chromatography or by high performance liquid chromatography. SDS gel electrophoresis data indicate that the SP-B samples obtained using different solvents showed different oligomerization states of the protein. The CD and FTIR spectra of SP-B isolated from all extracts were consistent with a secondary structure dominated by a-helix. The CD and FTIR spectra of the first SP-C corresponded to an a-helical secondary structure and the spectra of the second SP-C corresponded to a mixture of a-helical and b-sheet conformation. In contrast, the spectra of the third SP-C corresponded to antiparallel b-sheets. The interfacial behavior was characterized by surface pressure/area (p-A) isotherms. Differences in the oligomerization state of SP-B as well as in the secondary structure of SP-C all produce significant differences in the surface pressure/area isotherms. The molecular cross sections determined from the p-A isotherms and from dynamic cycling experiments were 6 nm 2 /dimer molecule for SP-B and 1.15 nm 2 /molecule for SP-C in a-helical conformation and 1.05 nm 2 /molecule for SP-C in b-sheet conformation. Both the oligomer ratio of SP-B and the secondary structure of SP-C strongly influence organization and behavior of these proteins in monolayer assemblies. In addition, a-helix ! b-sheet conversion of SP-C occurs simply by an increase of the summary protein/lipid concentration in solution.
Biophysical Journal, 1994
Spread binary monolayers of surfactant-associated proteins SP-B and SP-C were formed at the air-water interface. Surface pressure measurements showed no interactions between the hydrophobic proteins. The effects of a mixture of SP-B plus SP-C (2:1, w/w) on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and DPPC:DPPG (7:3, mol:mol) were studied. During compression of ternary and quaternary films, containing less than 0.4 mol% or 5 weight% total protein, the proteins were not squeezed out and appeared to remain associated with the film until collapse at surface pressures of about 65-70 mN*m-1. At initial concentrations of total protein of about 0.9 mol% or 10 weight%, exclusion of protein-lipid complexes was observed at 40-50 mNm-1. Larger amounts of phospholipid were removed by proteins from (SP-B:SP-C)/DPPG films than from (SP-B:SP-C)/DPPC ones. Separate squeeze-out of SP-B (or SP-B plus DPPC) at about 40 mNm-1, followed by exclusion of SP-C (or SP-C plus DPPC) at about 50 mN-m-1, was observed in (SP-B:SP-C)/DPPC films. This led to a conclusion that there was independent behavior of SP-B and SP-C in (SP-B:SP-C)/DPPC monolayers. The quaternary (SP-B:SP-C)/(DPPC:DPPG) films showed qualitatively similar process of squeeze-out of the proteins. In the ternary mixtures of SP-B plus SP-C with DPPG separate exclusion of SP-B was not detected; rather, the data was consistent with exclusion of a (SP-B:SP-C)/DPPG complex at about 50 mN*m-1. The results imply possible interactions between SP-B and SP-C and the acidic phospholipid.