Deciphering structure and topology of conserved COG2042 orphan proteins.
BACKGROUND
The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon Sulfolobus solfataricus was purified to homogeneity for biophysical characterization.


RESULTS
The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry-based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region Glu73-Arg78 is hyper-sensitive, and thus accessible and flexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 amino acid 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 cross-linked. These experimental data have been used to rank multiple three-dimensional models generated by a de novo procedure.


CONCLUSIONS
Our data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs.
Armengaud
Jean
J
CEA-VALRHO, DSV-DIEP-SBTN, Service de Biochimie post-génomique & Toxicologie Nucléaire, Bagnols-sur-Cèze, France. armengaud@cea.fr
Dedieu
Alain
A
Solques
Olivier
O
Pellequer
Jean-Luc
JL
Quemeneur
Eric
E
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D001710:Biotin / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry; D048229:Sulfolobus solfataricus / Q000378:metabolism*
D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; D010455:Peptides; D011994:Recombinant Proteins; D013388:Succinimides; D018698:Glutamic Acid; D001710:Biotin; D001120:Arginine; D010447:Peptide Hydrolases; D008239:Lysine; C001426:N-hydroxysuccinimide