1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus

Abstract

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.

Lipid-Free Antigen B Subunits from Echinococcus granulosus: Oligomerization, Ligand Binding, and Membrane Interaction Properties

“…Our next step was to identify a method to efficiently remove bacterial ligands from the proteins. Despite published precedent [ 35 ], we found that chromatography with Lipidex 1000 is ineffective at removing lipid from helminth LBPs [ 39 , 57 , 58 ]. We therefore exploited a method for removal of co-purifying ligands from other recombinant LBPs produced in E .…”

Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

“…Recombinant Na-FAR-1 (rNa-FAR-1) was expressed in BL21 (λDE3) Escherichia coli cells as described [ 27 ]. For native crystallographic studies, Na-FAR-1 was purified to homogeneity, as previously described [ 28 ], from cells grown in LB media.…”

“…For NMR studies, samples of unlabelled, 15 N-labelled and 13 C 15 N-labelled protein were purified by nickel-affinity, size exclusion and reverse-phase chromatographies, as described [ 27 ], from cells grown in M9 minimal medium containing 15 NH 4 Cl, [ 13 C 6 ]-glucose or their unlabelled equivalents.…”

“…Apo -Na-FAR-1 samples were delipidated by reverse phase-HPLC (high performance liquid chromatography) as previously described [ 27 ] using a C 8 -silica stationary phase and a water/acetonitrile gradient in the presence of 0.1% trifluoroacetic acid as the mobile phase. Samples were lyophilized, reconstituted and concentrated to approximately 0.6 mM in 20 mM sodium phosphate, pH 7.20.…”

“…All spectra were recorded at 311 K on a Bruker AVANCE 600 MHz spectrometer equipped with TCI cryoprobe. Resonances were assigned, as described previously [ 27 ]. All spectra were processed in AZARA (Wayne Boucher, http://www.bio.cam.ac.uk/azara ).…”