Biocomputational studies on protein structures

Sammanfattning: Biology in the post-genomic era produces large amount of data. This, in combination with the need for efficient algorithms to find genes in the genomic material, has brought a renaissance into the field of computational biology. Methods now range from lead discovery in the drug discovery process, by virtual ligand screening, through sequence comparisons and homology searches, to micro array data analysis and visualisation. This thesis primarily deals with sequence analysis, different aspects of protein structure prediction and ligand--enzyme complex characterisation, I have applied bioinformatic techniques on the enzyme families of medium- chain dehydrogenases/reductases (MDR), short-chain dehydrogenases/reductases (SDR) and biologically active peptides Sequence analysis of the MDR superfamily extends the evolutionary context of this superfamily, as MDR enzymes were collected from completely sequenced genomes. The analysis reveals the presence of eight families whereof several were previously uncharacterised. Three families are formed by dimeric alcohol dehydrogenases (ADH), cinnamyl alcohol dehydrogenases (CAD) and tetrameric alcohol dehydrogenases (YADH). Three further families are centred on forms initially detected as mitochondrial respiratory function proteins (MRF), acetyl-CoA reductases of fatty acid synthases (ACR), and leukotriene B4 dehydrogenases (LTD). The two remaining families, with polyol dehydrogenases (PDH) and quinone reductases (QOR), are also distinct but with variable sequences. The analysis also suggests that new functions have evolved in this superfamily in higher organisms. Factors that govern the substrate specificity of gammagammaADH were investigated with docking calculations and can be traced to active site characteristics, most notably the Ser48/thr48 replacement between gammagammaADH and betabetaADH, which allow the oxidation of 3beta-hydroxy bile acids, such as isoUDCA, in gammagammaADH, while both enzymes are inactive versus 3alpha-hydroxy bile acids. A homology model of type 10 17beta-hydroxysteroid dehydrogenase was constructed from 7alpha-hydroxysteroid dehydrogenase. The validity of the model was investigated by its ability to distinguish between active and inactive using docking calculations. Substrates tested ranged from steroids and bile acids to L- and D-hydroxyacyl CoA. Ligands with 17P or 3alpha hydroxy groups and L-hydroxyacyl CoA could achieve interactions favourable for catalysis at the active site. A crystallographically determined structure published after the submission of our paper verified large portions of our model. The role of a conserved asparagine in the short-chain dehydrogenase/reductase (SDR) fold is investigated through structural comparisons of 21 members with experimentally verified structures. An extensive hydrogen-bonding network including parts of the active site is revealed in 16 out of 21 SDR forms. Molecular dynamics simulations were employed to study the effect of deleterious mutants and replacements, known to diminish fibrillation, to the stability of the helical region of the amyloidogenic peptides amyloid beta-peptide (AP) and surfactant protein-C (SP-C). The effects in SP-C are quantitatively distinguishable, while the noise ratio in the AP simulations makes valid predictions somewhat difficult. Sequence comparisons of the C-peptide of proinsulin displays a sequence variability that is 1 to 2 orders of magnitude greater than that of insulin, but in the same order of magnitude as the well established peptide hormones relaxin and parathormone, which in conjunction with functional reports may indicate a hormonal function for the C-peptide.