Experimental annotation of post-translational features and translated coding regions in the pathogen Salmonella Typhimurium

<p>Abstract</p> <p>Background</p> <p>Complete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. However, determining protein-coding genes for most new genomes is almost completely performed by inference using computational predictions with significant documented error rates (> 15%). Furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function.</p> <p>Results</p> <p>We experimentally annotated the bacterial pathogen <it>Salmonella </it>Typhimurium 14028, using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. The data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in <it>Salmonella </it>and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. Additionally, we uncovered 12 non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel ORFs in <it>Salmonella </it>pathogenesis. We also characterized post-translational features in the <it>Salmonella </it>genome, including chemical modifications and proteolytic cleavages. We find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. Our <it>in vivo </it>proteolysis data identified more than 130 signal peptide and N-terminal methionine cleavage events critical for protein function.</p> <p>Conclusion</p> <p>This work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of <it>Salmonella </it>as a resource for systems analysis.</p

X-ray studies on crystalline complexes involving amino acids and peptides. XXXV. Invariance and variability in amino acid aggregation in the complexes of maleic acid with L-histidine and L-lysine

The crystal structures of complexes of maleic acid with l-histidine and l-lysine have been determined. The two crystallographically independent amino acid molecules in the l-histidine complex have different closed conformations, while the lysine molecule in its complex has the most favourable conformation sterically with an all-trans sidechain trans to the -carboxylate group. The maleic acid molecules exist as semimaleate ions of similar conformation and contain a symmetric O H O hydrogen bond. Amino acid cations and semimaleate anions aggregate into alternate layers in both the structures. The arrangement of molecules in the histidine layer in l histidine semi- maleate is closer to that in the crystals of the free amino acid than in other l-histidine complexes. On the other hand, the arrangement of lysine molecules in its semi-maleate complex is different from any observed so far. However, the well established characteristic interaction patterns involving amino and carboxylate groups still play a major role in holding the molecules together in the crystal of the complex

The combination of molecular dynamics with crystallography for elucidating protein–ligand interactions: a case study involving peanut lectin complexes with T-antigen and lactose

Peanut lectin binds T-antigen $[Gal\beta (1–3)GalNAc]$ with an order of magnitude higher affinity than it binds the disaccharide lactose. The crystal structures of the two complexes indicate that the higher affinity for T-antigen is generated by two water bridges involving the acetamido group. Fresh calorimetric measurements on the two complexes have been carried out in the temperature range 280–313K. Four sets of nanosecond molecular-dynamics(MD) simulations, two at 293K and the other two at 313K, were performed on each of the two complexes. At each temperature, two some what different protocols were used to hydrate the complex in the two runs. Two MD runs under slightly different conditions for each complex served to assess the reliability of the approach for exploring protein–ligand interactions. Enthalpies based on static calculations and on MD simulations favour complexation involving T-antigen. The simulations also brought to light ensembles of direct and water-mediated protein–sugar interactions in both the cases. These ensembles provide a qualitative explanation for the temperature dependence of the thermodynamic parameters of peanut lectin–T-antigen interaction and for the results of one of the two mutational studies on the lectin. They also support the earlier conclusion that the increased affinity of peanut lectin for T-antigen compared with that for lactose is primarily caused by additional water bridges involving the acetamido group. The calculations provide a rationale for the observed sugar-binding affinity of one of the two available mutants. Detailed examination of the calculations point to the need for exercising caution in interpreting results of MD simulations: while long simulations are not possible owing to computational reasons, it is desirable to carry out several short simulations with somewhat different initial conditions

Crystal Structures of Artocarpin, a Moraceae Lectin with Mannose Specificity, and its Complex with Methyl-alpha-D-mannose: Implications to the Generation of Carbohydrate Specificity

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the b-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-a-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the speciÆcities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold

Crystallization and preliminary X-ray studies of snake gourd lectin: homology with type II ribosome-inactivating proteins

The lectin from the seeds of snake gourd (Trichosanthes anguina) has been crystallized in two forms using the hanging-drop method. Both the forms are hexagonal, with the asymmetric unit containing one subunit consisting of two polypeptide chains linked through disulfide bridges. Intensity data from one of the forms were collected at room temperature as well as at low temperature to 3 A resolution. Molecular-replacement studies indicate that the lectin is homologous to type II ribosome-inactivating proteins. Partial refinement confirms this conclusion

Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose

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