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Role of the N-terminal seven residues of surfactant protein B (SP-B).

ABSTRACT: Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state (2)H NMR were used to study the structure of SP-B (1-25,63-78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8-25,63-78) and SP-B (1-25,63-78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics.

SUBMITTER: Sharifahmadian M

PROVIDER: S-EPMC3759391 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Role of the N-terminal seven residues of surfactant protein B (SP-B).

Sharifahmadian Mahzad M Sarker Muzaddid M Palleboina Dharamaraju D Waring Alan J AJ Walther Frans J FJ Morrow Michael R MR Booth Valerie V

PloS one 20130902 9

Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78), also cal ...[more]

PMID: 24023779

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Project description: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.

Project description:Natural surfactant preparations containing phospholipids and the hydrophobic surfactant proteins B and C (SP-B and SP-C) are effective in the treatment of respiratory distress syndrome in premature infants. The limited supply, and the risk of infectious agents and immunological reactions have promoted the evaluation of synthetic peptides in surfactant preparations. However, the folding of synthetic SP-C into an alpha-helix is inefficient and alpha-helical SP-C analogues with Val-->Leu substitutions form oligomers. In order to circumvent these problems we have synthesized an SP-C analogue, named SP-C(LKS), which differs from SP-C mainly by the exchange of most of the Val residues in positions 16-28 with Leu residues to promote an alpha-helical conformation, and by the introduction of Lys residues at positions 17, 22 and 27 in order to locate positive charges around the helical circumference and thereby avoid self polymerization. CD spectroscopy showed a spectrum typical for alpha-helical peptides and SDS/PAGE disclosed a single band. The biophysical activity of artificial surfactant preparations containing SP-C(LKS) and phospholipids, with and without native SP-B, was measured using a Wilhelmy balance and a pulsating bubble surfactometer. SP-C(LKS) (3%, w/w) in a mixture of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol/palmitic acid (68:22:9, by wt.) suspended in 150 mM NaCl, showed rapid spreading at the air-liquid interface and produced a surface tension of <1 mN/m at minimum bubble size (gammamin) and 42 mN/m at maximum bubble size (gammamax) in the pulsating bubble surfactometer. The addition of 2% (w/w) SP-B to the preparation reduced the maximum surface tension to 33-35 mN/m, i.e. both gammamin and gammamax values were similar to those of natural surfactant preparations. Optimal in vitro characteristics were also obtained from a preparation containing SP-C(LKS), SP-B, DPPC and phosphatidylglycerol, i.e. when palmitic acid was omitted from the lipid mixture. SP-B containing surfactant preparations made up in Hepes buffer at pH 6.9, instead of in 150 mM NaCl, had similar biophysical activity provided that palmitic acid was omitted, but decreased activity in the presence of palmitic acid.

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Role of the N-terminal seven residues of surfactant protein B (SP-B).

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Role of the N-terminal seven residues of surfactant protein B (SP-B).

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