Construction d'un site internet d'aide à la détermination des espèces de champignons les plus fréquemment rencontrées dans le Nord de la France

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Deciphering structure and topology of conserved COG2042 orphan proteins

Background: The cluster of orthologous group COG2042 has members in all sequencedEukaryota as well as in many Archaea. The cellular function of these proteins of ancient originremains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-relatedproteins that have been functionally or structurally characterized. As a prototype among COG2042orthologs, SSO0551 protein from the hyperthermophilic archaeon Sulfolobus solfataricus waspurified to homogeneity for biophysical characterization.Results: The untagged protein is thermostable and behaves as a monomeric protein in gel filtrationexperiment. Several mass spectrometry-based strategies were combined to obtain a set of lowresolution structural information. Kinetic data from limited proteolysis with various endoproteasesare concordant in pointing out that region Glu73-Arg78 is hyper-sensitive, and thus accessible andflexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 aminoacid RLI motif at the N terminus is solvent exposed. Cross-links between Lys10-Lys14 and Lys23-Lys25 indicate that these residues are spatially close and in adequate conformation to be crosslinked. These experimental data have been used to rank multiple three-dimensional modelsgenerated by a de novo procedure.Conclusion: Our data indicate that COG2042 proteins may share a novel fold. Combiningbiophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structuralinformation and to help in prioritizing targets in structural genomics program

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-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)

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-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