Selective Labeling of Pulmonary Surfactant Protein SP-C in Organic Solution

Plasencia, I; Cruz, Antonio; López-Lacomba, José Luís; Casals, Cristina; Pérez‐Gil, Jesús

doi:10.1006/abio.2001.5222

Cited by 15 publications

(14 citation statements)

References 40 publications

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“…We have previously shown that the dansylated form of SP-C used in this work maintains its acylation state and secondary structure as native SP-C, as well as the ability to promote interfacial phospholipid adsorption and to perturb the thermotropic properties of DPPC bilayers [31]. The analysis of the fluorescent properties of Dns-SP-C in phospholipid bilayers can, therefore, provide information on lipid-protein and proteinprotein interactions potentially occurring with native SP-C.…”

Section: Discussionmentioning

confidence: 90%

“…Labelling of SP-C with the derivative Dns-ITC was carried out as described previously [31]. Briefly, the apparent pH of a solution containing approx.…”

Section: Protein Preparationmentioning

confidence: 99%

“…Dns-SP-C bears a single dansyl group at its N-terminal end and conserves its two cysteine residues palmitoylated. The modified protein conserves the secondary structure of native SP-C, as well as its ability to promote interfacial adsorption of DPPC suspensions and to influence the thermotropic behaviour of DPPC bilayers [31]. The dansylated derivative has been also used to find out whether SP-C interacts with SP-B in phospholipid bilayers, through the analysis of fluorescence resonance energy transfer (RET) from the single SP-B tryptophan residue to the SP-C dansyl fluorophore.…”

Section: Introductionmentioning

confidence: 99%

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Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B‒SP-C interactions in phospholipid bilayers

et al. 2001

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A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), bearing a single dansyl group at its N-terminal end, has been used to characterize the lipid-protein and protein-protein interactions of SP-C reconstituted in phospholipid bilayers, using fluorescence spectroscopy. The fluorescence emission spectrum of Dns-SP-C in phospholipid bilayers is similar to the spectrum of dansyl-phosphatidylethanolamine, and indicates that the N-terminal end of the protein is located at the surface of the membranes and is exposed to the aqueous environment. In membranes containing phosphatidylglycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with respect to the fluorescence of phosphatidylcholine (PC), suggesting that electrostatic lipid-protein interactions induce important effects on the structure and disposition of the N-terminal segment of the protein in these membranes. This effect saturates above 20% PG molar content in the bilayers. The parameters for the interaction of Dns-SP-C with PC or PG have been estimated from the changes induced in the fluorescence emission spectrum of the protein. The protein had similar K(d) values for its interaction with the different phospholipids tested, of the order of a few micromolar. Cooling of Dns-SP-C-containing dipalmitoyl PC bilayers to temperatures below the phase transition of the phospholipid produced a progressive blue-shift of the fluorescence emission of the protein. This effect is interpreted as a consequence of the transfer of the N-terminal segment of the protein into less polar environments that originate during protein lateral segregation. This suggests that conformation and interactions of the N-terminal segment of SP-C could be important in regulating the lateral distribution of the protein in surfactant bilayers and monolayers. Potential SP-B-SP-C interactions have been explored by analysing fluorescence resonance energy transfer (RET) from the single tryptophan in porcine SP-B to dansyl in Dns-SP-C. RET has been detected in samples where native SP-B and Dns-SP-C were concurrently reconstituted in PC or PG bilayers. However, the analysis of the dependence of RET on the protein density excluded specific SP-B-Dns-SP-C associations.

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Section: Discussionmentioning

confidence: 90%

“…Labelling of SP-C with the derivative Dns-ITC was carried out as described previously [31]. Briefly, the apparent pH of a solution containing approx.…”

Section: Protein Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B‒SP-C interactions in phospholipid bilayers

et al. 2001

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“…Fluorescently labeled surfactant proteins were prepared by labeling purified SP-B and SP-C solutions in organic solvent, as described previously (26). This procedure allows attachment of a single fluorophore to the N-terminal amine group of the protein.…”

Section: Methodsmentioning

confidence: 99%

Cholesterol Rules

Serna¹,

Pérez‐Gil²,

Simonsen³

et al. 2004

Journal of Biological Chemistry

266

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Pulmonary surfactant, the lipid-protein material that stabilizes the respiratory surface of the lungs, contains approximately equimolar amounts of saturated and unsaturated phospholipid species and significant proportions of cholesterol. Such lipid composition suggests that the membranes taking part in the surfactant structures could be organized heterogeneously in the form of inplane domains, originating from particular distributions of specific proteins and lipids. Here we report novel results concerning the lateral organization of bilayer membranes made of native pulmonary surfactant where the coexistence of two distinct micrometer sized fluid phases (fluid ordered and fluid disordered-like phases) is observed at physiological temperatures by using fluorescence microscopy and atomic force microscopy. Additional experiments using fluorescent-labeled proteins SP-B and SP-C show that at physiological temperatures these hydrophobic proteins are located exclusively in the fluid disordered-like phase. Most interestingly, the microscopic coexistence of fluid phases is maintained up to 37.5°C, where most fluid ordered phases melt. This observation suggests that the particular composition of this material is naturally designed to be at the "edge" of a lateral structure transition under physiological conditions, likely providing particular structural and dynamic properties for its mechanical function. The observed lateral structure in native pulmonary surfactant membranes is dramatically affected by the extraction of cholesterol, an effect not observed upon extraction of the surfactant proteins. Furthermore, the spreading properties of the native surfactant material at the air-liquid interface were also greatly affected by cholesterol extraction, suggesting a connection between the observed lateral structure and a physiologically relevant function of the material. We suggest that the particular lipid composition of surfactant could be finely tuned to provide, under physiological conditions, a structural scaffold for surfactant proteins to act at appropriate local densities and lipid composition.

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“…A major problem is that most of the physico-chemical features of SP-C are dominated by contributions from the hydrophobic α-helix, making it difficult to assign relevant properties originating from the N-terminal segment of the protein. Lipidprotein interactions involving this region of SP-C have been studied previously by using protein forms that were chemically derivatized to introduce extrinsic probes at the N-terminal end [22,23]. Those studies indicated that the N-terminal segment of SP-C is located at the surface of phospholipid bilayers, exposed to the aqueous environment.…”

Section: Introductionmentioning

confidence: 99%

The N-terminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers

Plasencia

Rivas

Keough

et al. 2004

Biochemical Journal

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In the present study, 13-residue peptides with sequences corresponding to the native N-terminal segment of pulmonary SP-C (surfactant protein C) have been synthesized and their interaction with phospholipid bilayers characterized. The peptides are soluble in aqueous media but associate spontaneously with bilayers composed of either zwitterionic (phosphatidylcholine) or anionic (phosphatidylglycerol) phospholipids. The peptides show higher affinity for anionic than for zwitterionic membranes. Interaction of the peptides with both zwitterionic and anionic membranes promotes phospholipid vesicle aggregation, and leakage of the aqueous content of the vesicles. The lipid-peptide interaction includes a significant hydrophobic component for both zwitterionic and anionic membranes, although the interaction with phosphatidylglycerol bilayers is also electrostatic in nature. The effects of the SP-C N-terminal peptides on the membrane structure are mediated by significant perturbations of the packing order and mobility of phospholipid acyl chain segments deep in the bilayer, as detected by differential scanning calorimetry and spin-label ESR. These results suggest that the N-terminal region of SP-C, even in the absence of acylation, possesses an intrinsic propensity to interact with and perturb phospholipid bilayers, thereby potentially facilitating SP-C promoting bilayer-monolayer transitions at the alveolar spaces.

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Selective Labeling of Pulmonary Surfactant Protein SP-C in Organic Solution

Cited by 15 publications

References 40 publications

Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B‒SP-C interactions in phospholipid bilayers

Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B‒SP-C interactions in phospholipid bilayers

Cholesterol Rules

The N-terminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers

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