Deciphering structure and topology of conserved COG2042 orphan proteins-3

<p><b>Copyright information:</b></p><p>Taken from "Deciphering structure and topology of conserved COG2042 orphan proteins"</p><p>BMC Structural Biology 2005;5():3-3.</p><p>Published online 8 Feb 2005</p><p>PMCID:PMC549553.</p><p>Copyright © 2005 Armengaud et al; licensee BioMed Central Ltd.</p>ith a trypsin/SSO0551 protein ratio of 1/20 (w/w). The products were then resolved onto a C8 reverse phase chromatographic column and the different UV absorbing fractions were analyzed by MALDI-TOF. The spectrum obtained with the fraction eluting at 40–50 % acetonitrile is shown. The asterisk labels a peak arising from trypsin autolysis. Peaks that could be assigned are identified with numbers (experimental , residues, Δmass in ppm compared to theoretical [M+H]average mass): (19198.4, [1–166], -1), (15478.7, [32–166], -53), (15191.2, [35–166], +153), (12883.4, [52–162] or [56–166], -46 or -43), (12724.2, [57–166], +193), (10603.8, [76–166], +51), (10220.1, [79–166], +77), (9257.2, [83–162], +63) and (7717.0, [101–166], -134). Peptides and , complementary of and , were not observed in this spectrum but in another fraction from the C8 reverse-phase chromatography corresponding to smaller peptides

Deciphering structure and topology of conserved COG2042 orphan proteins-0

<p><b>Copyright information:</b></p><p>Taken from "Deciphering structure and topology of conserved COG2042 orphan proteins"</p><p>BMC Structural Biology 2005;5():3-3.</p><p>Published online 8 Feb 2005</p><p>PMCID:PMC549553.</p><p>Copyright © 2005 Armengaud et al; licensee BioMed Central Ltd.</p>Archaeal and eukaryal homologs were obtained from public databases [54] by PSI-BLAST searches. To get the most conserved alignment between COG2042 polypeptide sequences, most probable start codons should be considered as atg at nucleotide 500978 on Crick strand for MTH554 from str. DeltaH (NC_000916), gtg at nucleotide 1526308 on Watson strand for Vng2075c from sp. NRC-1 (NC_002607), atg at nucleotide 8398 on Watson strand for Mbur141901 from DSM6242 (NZ_AADH01000008) and atg at nucleotide 484790 on Crick strand for SSO0551 from (NC_002754). Asterisks indicate modified protein sequences according to this new proposed annotation. Multiple alignments were performed by ClustalW. Following removal of a few ambiguously aligned regions, a data set was assembled comprising 40 sequences over 162 amino acid positions. An unrooted evolutionary distance tree was constructed based on Kimura distances and neighbor joining tree reconstruction algorithm. Bootstrap confidence levels at nodes were computed by the Phylips package with 400 replicates. Scale bar represents unit of amino acid substitutions per position. Accession numbers (gi) are indicated beside the organism. B – Conserved sequence blocks in the alignment of COG2042 members. Based on the phylogenetic analysis (Panel A), eight representative sequences were selected out of 40 COG2042 sequences. Four first sequences are from Archaea while last four sequences are from Eukaryota. SSO0551 sequence (gi38605619) from , labeled with an asterisk, has been numbered according to the new annotation proposed in RESULTS section. Invariant residues in the eight sequences are shown in red and conserved residues in blue. Two conserved motifs commented in the results section are indicated with grey boxes

Deciphering structure and topology of conserved COG2042 orphan proteins-2

<p><b>Copyright information:</b></p><p>Taken from "Deciphering structure and topology of conserved COG2042 orphan proteins"</p><p>BMC Structural Biology 2005;5():3-3.</p><p>Published online 8 Feb 2005</p><p>PMCID:PMC549553.</p><p>Copyright © 2005 Armengaud et al; licensee BioMed Central Ltd.</p>mposition of SSO0551 recombinant product

Deciphering structure and topology of conserved COG2042 orphan proteins-4

<p><b>Copyright information:</b></p><p>Taken from "Deciphering structure and topology of conserved COG2042 orphan proteins"</p><p>BMC Structural Biology 2005;5():3-3.</p><p>Published online 8 Feb 2005</p><p>PMCID:PMC549553.</p><p>Copyright © 2005 Armengaud et al; licensee BioMed Central Ltd.</p>at room temperature. Unlabeled protein mass spectrum is shown in grey solid line. Reagent/total lysine ratios were in Fig. 5A: 1:40 (black dotted line) and 1:20 (black dashed line), and in Fig. 5B: 1:2 (black solid line) and 2:1 (grey dotted line). The number of labeled lysines is indicated above each peak

Deciphering structure and topology of conserved COG2042 orphan proteins-5

<p><b>Copyright information:</b></p><p>Taken from "Deciphering structure and topology of conserved COG2042 orphan proteins"</p><p>BMC Structural Biology 2005;5():3-3.</p><p>Published online 8 Feb 2005</p><p>PMCID:PMC549553.</p><p>Copyright © 2005 Armengaud et al; licensee BioMed Central Ltd.</p>a molar ratio 1:20 (protein:DTSSP). They were then subjected to trypsin proteolysis. Masses of peptides treated with DTT (grey spectrum) or untreated (black spectrum) were measured by MALDI-TOF mass spectrometry. Peptide sequences are indicated and DTSSP cross-links or DTSSP moieties arising from DTT treatment are depicted schematically. Monoisotopic masses of protonated peptides [MH] are theoretically: 1491.807 amu and 1493.822 amu for LVKLKIAEFTR [21-31] cross-linked with one DTSSP molecule (untreated (CHNOS) and DTT-reduced (CHNOS), respectively), 1835.846 amu and 1837.862 amu for VYIIDYHKDDPKR [3-15] cross-linked with one DTSSP molecule (untreated (CHNOS) and DTT-reduced (CHNOS), respectively)

Biacore experiment to evaluate the reactivity between scFv 2LF and the five LF variants, in which H229, R230, Q234, L235 or Y236 had been individually mutated in alanine, or the LF variant where these five residues had been simultaneously mutated in alanine.

<p>A CM5 chip was sensitized with scFv 2LF. LF (control, in grey) and its five variants in which H229 (yellow), R230 (blue), Q234 (red), L235 (green) or Y236 (brown) had been individually mutated in alanine reacted in flux. The LF variant in which these five positions had been simultaneously mutated in alanine is presented in blue. Dilutions of LF and its variants giving an equivalent maximal signal are presented, to allow the comparison of the dissociation phases (after the maximal signal and the artefactual spikes) when dissociation constants are measured (between the 500^th and 700^th seconds). Values were calculated on several curves for each variant, and are presented on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone-0065855-t002" target="_blank">table 2:</a> the five variants with punctual mutations present at least a three-fold more rapid dissociation than the control, and the variant with the five simultaneous mutations presents no reactivity.</p

Biacore experiment to evaluate the reactivity between anti-LF polyclonal antibodies and the two LF variants, in which LF(178–184) or LF (231–236) had been shaved in alanine.

<p>A CM5 chip was sensitized using LF variants in which LF(178–184) (blue) or LF (231–236) (red) had been shaved in alanine, as these variants did not react with scFv 2LF and their presence and general conformation had to be verified. The diluted (1:100) serum of a macaque, drawn before immunization with LF,was used as a negative control and did not react with these two LF variants (dashed curves). The serum from the same animal after hyper-immunization with LF reacted with these two LF variants (continuous curves), showing that they had been immobilized on the CM5 chip. The curves are representative of two separate Biacore experiments, realized in the same conditions except for the variants used for sensitization.</p

IgG 2LF inhibits the formation of edema induced by the edema toxin (ET).

<p>(A) Photographs taken 12 hours after injection in mouse footpads of 20 µg ET (left picture), or of 20 µg ET pre-mixed with 5 µg (middle picture) or with 10 µg (right picture) of IgG 2LF. Ten µg of IgG 2LF premixed with 20 µg ET completely inhibited edema formation, whereas 5 µg of IgG 2LF reduced both the size and the duration of the edema; 2 µg of IgG 2LF had no appreciable effect. (B) Evolution of the sizes (dorsal/plantar) of mouse footpads after injection of 20 µg ET (blue), or of 20 µg ET pre-mixed with 2 µg (red) or 5 µg (yellow) or 10 µg (light blue) of IgG 2LF. Injection of PBS only (purple) was used as a negative control. Curves represent triplicate measurements for each timepoint. Pre-mixing with 10 µg of IgG 2LF completely abolished the formation of edema by 20 µg ET, and pre-mixing with 5 µg of IgG 2LF limited the size of the edema and reduced its duration. Pre-mixing of 20 µg ET with 2 µg of IgG 2LF had no appreciable effect.</p

Biacore experiment to measure the affinity of scFv 2LF for EF.

<p>Sensorgrams were obtained at EF concentrations of 220 nM (red curve), 110 nM (light blue), 55 nM (dark blue), 28 nM (brown) and 14 nM (green) on a CM5 chip sensitized by 250 response units of scFv 2LF. The affinity of scFv 2LF for EF was measured as 5 nM.</p

Evaluation of the reactivity of scFv 2LF with EF by ELISA and western blot.

<p>A/ Reactivity of scFv 2LF with EF in ELISA. Reactivity of scFv 2LF with EF is represented in blue, and LF (red) and KLH (green) were used as positive and negative controls, respectively. ScFv 2LF reacted with EF, though less strongly than with LF at high scFv concentrations. B/ Reactivity of scFv 2LF with EF in western blots under reducing conditions (lower part). EF (5, 2 and 1 µg/ml in lanes 4, 5 and 6, respectively) and LF as a positive control (5, 2 and 1 µg/ml in lanes 1, 2 and 3, respectively) were separated by SDS-PAGE electrophoresis (upper part) and their sizes verified to be 90 kDa by separation under reducing conditions. ScFv 2LF reacted similarly with EF and LF under reducing conditions.</p