Fig 1.
Amino acid sequence analysis of EgAgB8 subunits.
Analysis of EgAgB8 subunits were undertaken using Biology Workbench 3.2 (University of Illinois, National Center for Supercomputing Applications, USA). (A) Alignment of amino acid sequences of the mature peptides of EgAgB8/1, EgAgB8/2, EgAgB8/3, EgAgB8/4 and EgAgB8/5, using ClustalW from Biology Workbench 3.2. Absolutely conserved residues, partially residues and similar residues are shown in black, dark grey and light grey, respectively. Comparison between EgAgB8 mature peptides shows that EgAgB8/1, EgAgB8/3 and EgAgB8/5 are more closely related to each other than to EgAgB8/2 and EgAgB8/4, as shown in the alignment (B) and in the tree (C) obtained employing DrawTree from Biology Workbench 3.2. (D) Biochemical data for EgAgB8/2 and EgAgB8/3 (from sequences squared in A). The theoretical isoelectric point (pI) and the theoretical extinction coefficient were also estimated using Biology Workbench 3.2 software. Accession numbers: EgAgB8/1: AAD38373, EgAgB8/2: AAC47169, EgAgB8/3: AAK64236, EgAgB8/4: AAQ74958 and EgAgB8/5: BAE94835.
Fig 2.
Removal of co-purifying hydrophobic ligands from rEgAgB8 by RP-HPLC.
The lipid composition of rEgAgB8 subunits prior to delipidation (Pre), as well as rEgAgB8 subjected to RP-HPLC (Post) were analysed by TLC, in parallel with standards for neutral and polar lipids. Lipid bands were visualised using CuSO4/H3PO4 and identified by comparison with the standards. (A) The lipids extracted from E. coli grown under the same culture conditions is shown for comparison. TLCs from neutral and polar lipids were undertaken separately. (B) The lipid moiety of recombinant subunits were analysed by TLC using double development. The lipid fraction of rEgAgB8 pre-HPLC contained mainly polar lipids (PE and CL), which were successfully removed by the RP-HPLC method. PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; CL: cardiolipin; PE: phosphatidylethanolamine; Cho: cholesterol; FA: free fatty acids; DAG: diacylglycerols; TAG: triacylglycerols; SE: sterol esters.
Fig 3.
Far UV CD-spectra of lipid-free EgAgB8 subunits.
After purification and delipidation, rEgAgB8/2 and rEgAgB8/3 were analysed by CD for secondary structural content. Both subunits showed a predominant alpha-helical structure with double minima at 208 and 222 nm in agreement with their predicted structures [14,33,59]. One representative experiment of two is shown for both EgAgB8 subunits.
Fig 4.
SEC analysis of lipid-free EgAgB subunits.
Analysis of EgAgB lipid-free subunits by SEC was undertaken on a Superdex 200 column employing a flow rate of 0.5 mL/min. Elution profiles of lipid-free EgAgB8/2 and EgAgB8/3 were followed at 215 nm. Both subunits showed a well-defined peak, with a molecular weight estimation of 62 kDa for EgAgB8/2 and 38 kDa for EgAgB8/3 according to standard proteins analysed under the same conditions (a: BSA, 66 kDa; b:carbonic anhydrase, 29 kDa; and c: cytochrome c, 12 kDa).
Fig 5.
Cross-linking of rEgAgB subunits with EDC.
Covalent cross-linking of proteins with EDC was carried out for 30 minutes at 25°C in PBS and separated on 15% SDS-PAGE followed by silver staining. Controls without added EDC were undertaken under the same conditions. (A) Cross-linking of lipid-free EgAgB8/2 and EgAgB8/3 subunits. (B) Cross-linking of non-delipidated EgAgB8/2 and EgAgB8/3 subunits. (C) Cross-linking of native EgAgB. (D) Control with lipid-free and non-delipidated ABA-1-A1, a recombinant single unit of the helix-rich LBP of the nematode parasite Ascaris suum, which is monomeric at the concentration used. Molecular masses of the standard proteins are indicated in each panel.
Fig 6.
Fluorimetric titration of 12-AS with EgAgB8 subunits.
Changes in relative 12-AS fluorescence were monitored from 400 to 500 nm after excitation at 383 nm upon incremental additions of EgAgB8/2 or EgAgB8/3 to a cuvette initially containing 2 mL of 0.5 μM 12-AS in TBS buffer. (A) Emission spectra of 12AS in TBS or upon adding EgAgB8/2 (0.5 μM). (B) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. (C) Emission spectra of 12AS in TBS or upon adding EgAgB8/3 (0.7 μM). (D) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. For both proteins, 12-AS spectra showed a blue shift in emission spectrum that accompanies a strong increase in fluorescence emission. The data were consistent with one binding site per monomer unit of protein and Kd values of 0.16 ± 0.09 μM for EgAgB8/2 and 0.34 ± 0.02 μM for EgAgB8/3 were obtained using SigmaPlot software. The solid line is the theoretical binding curve for complex formation. One representative experiment of three is shown for both EgAgB8 subunits.
Fig 7.
Equilibrium partition of 12-AS between EgAgB8 subunits and SUVs.
The Kp for 12-AS partitioning was determined by measuring 12-AS fluorescence after addition of SUVs into a solution containing 7.5 μM EgAgB8/2 or EgAgB8/3 and 0.5 μM 12-AS in buffer TBS at 25°C (15:1 mol:mol). (A), (B) Emission spectra changes of 12AS bound to EgAgB8/2 or EgAgB8/3 respectively, upon incremental addition of EPC-SUVs containing NBD-PC. (C), (D) Changes in relative 12-AS fluorescence were used to obtained Kp values, fitting the Equation 2 described in Materials and Methods to the data, employing Solver Add-In from Excel (solid line). Kp values of 0.62 ± 0.09 and 0.88 ± 0.15 were obtained for rEgAgB8/2 and rEgAgB8/3, respectively. One representative experiment of two is shown for both EgAgB8 subunits.
Fig 8.
Effect of acceptor membrane concentration on 12-AS transfer from EgAgB8/2 and EgAgB8/3 to different SUVs.
Transfer of 12-AS from EgAgB8/2 or EgAgB8/3 to SUVs was monitored by adding SUVs in a molar ratio of 10:1, 20:1 and 40:1 (SUVs/Protein) to the complex EgAgB8/2:12AS or EgAgB8/3:12AS (15:1 mol:mol). (A) Representative kinetic trace obtained when combining EgAgB8–12AS with NBD-PC-containing vesicles (molar ratio SUV/Protein of 10:1). Photobleaching control adding TBS instead of NBD-PC/SUVs is shown. (B) SUVs containing 100% EPC; (C) 75% EPC, 25% PS or (D) 75% EPC, 25% CL were used. For each experimental condition at least five replicates of the kinetic curves were done. All curves were well-described by a single exponential function to obtain each transfer rate value employing SigmaPlot software. Transfer rates (mean ± SD) of three independent experiments are reported. Statistical analysis of the data was carried out employing ANOVA followed by Tukey's Post Hoc Test from GraphPad Prism software.