Listragning i lerbruk

En undersökning rörande listdragning i lerbruk har utförts. Tre olika lerbruk utvärderades på testytor. Resultaten visade att alla tre bruken gav lister med skarpa linjer och släta ytor. Ett av bruken valdes sedan ut till listdragning på en uppmurad rörspis. Följande examensarbete innehåller bakgrund, en beskrivning av arbetet som utförts samt en sektion om resultat, ställningstaganden och diskussion.Uppsats för avläggande av högskoleexamen i Kulturvård, Bygghantverk,7,5 hp, 2011

Structural and functional studies on glutaredoxins and members of the thioredoxin superfamily

The active site of E.coli glutaredoxin 3 was investigated using a combination of experimental and theoretical techniques. Starting from different conformations, molecular dynamics simulations converged to an active site conformation where the Cys11 thiolate was hydrogen bonded to surrounding amide protons and to the thiol proton of Cys14. The Cys14 chi1 and chi2 torsion angles determined by NMR supported the simulation result. The torsion angles of Cys11 could not be determined due to degenerate β-protons. The NMR titration of His15 showed that it was the sidechain was in the ε tautomer form and had a pKa value of 6.0. The pH-induced unfolding of WT and the C14A and K8A mutants were monitored by UV- and CD-spectroscopy. The Cys11 thiolate becomes protonated when the protein unfolds, or the protein unfolds when Cys11 becomes protonated. The removal of the thiol group in the C14A had a large impact on the outcome of the titrations while the K8A mutant only had a marginal effect. Human spermatid-specific thioredoxin-1 is a protein that is expressed only in sperm during maturation. Analysis of crystallization trials showed that only one part of the protein was left. CD spectra of the full length protein and the two parts were collected. It was clear from the spectra of the N-terminal domain that it was largely unstructured. In contrast, the C-terminal domain was that of a folded protein and was found to be quite similar to human Trx1. There are two human dithol glutaredoxins. The redox potentials of these were determined using glutathione redox buffer, direct protein-protein equilibration and thermodynamic linkage. The values were found to be -232 mV and -221 mV for hGrx1 and hGrx2, respectively. Furthermore, a second disulfide bond was discovered. The redox potential was determined to be -317 mV. Hence, it is present as a disulfide in the protein except under very reducing conditions in vivo. Phylogenetic analysis showed that there are three distinct groups of glutaredoxins, Grx1, Grx2 and the monothiol Grx5. A high precision NMR structure of E.coli Grx3 was solved. The RMSD of the backbone atoms was 0.26 Å relative to the mean. The Cys11 residue in active site showed two conformations. In the first, the thiolate was the acceptor in a hydrogen bond network where the donors where the surrounding amide protons and the thiol proton of Cys14. The other conformation displayed a hydrogen bond between the Cys11 and Thr10. Double mutant cycle analyses showed that there is a favorable interaction between the side chains of Cys11 and Cys14 (-1.63 kcal/mol). However, the interaction between Thr10 and Cys11 was unfavorable (+0.68 kcal/mol). The structures of the oxidized form and of the Grx3-glutathione complex were recalculated in order to make a valid comparison to the reduced form. The analysis showed that there are small differences at the backbone level between the redox forms. There are some differences in the active site. The most apparent ones are the conformation of Tyr13 and the position of Val52, both important in substrate binding

Human spermatid-specific thioredoxin-1 (Sptrx-1) is a two-domain protein with oxidizing activity

24 páginas, 5 figuras.Spermatid-specific thioredoxin-1 (Sptrx-1) is the first member of the thioredoxin family of proteins with a tissue-specific expression pattern, found exclusively in the tail of elongating spermatids and spermatozoa. We describe here further biochemical characterization of human Sptrx-1 protein structure and enzymatic activity. In gel filtration chromatography human Sptrx-1 eluates as a 400 kDa protein consistent with either an oligomeric form, not maintained by intermolecular disulfide bonding, and/or a highly asymmetrical structure. Analysis of circular dichroism spectra of fragments 1–360 and 361–469 and comparison to spectra of full-length Sptrx-1 supports a two-domain organization with a largely unstructured N-terminal domain and a folded thioredoxin-like C-terminal domain. Functionally, Sptrx-1 behaves as an oxidant in vitro when using selenite, but not oxidized glutathione, as electron acceptor. This oxidizing enzymatic activity suggests that Sptrx-1 might govern the stabilization (by disulfide cross-linking) of the different structures in the developing tail of spermatids and spermatozoa.This work was supported by grants from the Swedish Medical Research Council (Projects 03P-14096-01A, 03X-14041-01A), the Åke Wibergs Stiftelse and the Karolinska Institutet to A.M.-V. and the Fundación Margit y Folke Pehrzon to A.J.Peer reviewe