“Hot standards” for the thermoacidophilic archaeon Sulfolobus solfataricus

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics

Structure of the complex of trypsin with a highly potent synthetic inhibitor at 0.97 Å resolution

The structure of the complex formed between bovine -trypsin and the highly potent synthetic inhibitor 2-{3'-[5'-methoxy-2'-(N-p-diaminomethylphenyl)amido]-1'-pyrido}-5-(N-2''-t-butylethanol)amidobenzoic acid (C<SUB>28</SUB>H<SUB>32</SUB>N<SUB>5</SUB>O<SUB>6</SUB>) has been determined at 0.97 Åresolution. X-ray intensity data were collected to 0.97 Å under cryocooled conditions. The structure was refined anisotropically using REFMAC5 and SHELX-97 to R<SUB>cryst</SUB> factors of 13.4 and 12.6% and R<SUB>free</SUB> factors of 15.7 and 16.3%, respectively. Several regions of the main chain and side chains that have not been previously observed were clearly defined in the present structure. H atoms are indicated as significant peaks in an |F<SUB>o</SUB> - F<SUB>c</SUB>| difference map, which accounts for an estimated 35% of all H atoms at the 2.5 σlevel. The C, N and O atoms are definitively differentiated in the electron-density maps. The amido part of the inhibitor occupies the specificity pocket and the remainder fills the remaining part of the ligand-binding cleft and interacts with the enzyme through an extensive network of hydrogen bonds. The inhibitor distorts the stereochemistry of the catalytic triad, Ser195-His57-Asp102, thereby blocking the proton-relay process of the active site by preventing the formation of the crucial hydrogen bond between His57 N<SUP>δ1</SUP> and Asp102 O<SUP>δ 1</SUP>