<p>Stabilization of proteins is of great interest for the biotechnological society, industrial as well as research areas. Proteins with high stability are more suitable as reagents, easier to handle, store, transport and use in industrial processes. One way to stabilize a protein is to introduce a disulfide bridge into the structure by protein engineering. In this report the formation of a disulfide bridge between engineered cysteines in non ideal conformations in human carbonic anhydrase has been investigated. The disulfide bridge is not formed when the protein is in its native state. It is shown that when the protein is exposed to mild concentrations of urea in the presence of DTTox the disulfide bridge is formed. Also upon refolding in vitro, in a non oxidative environment, disulfide bridges are formed. This observation is worth to notice, since the disulfide bridge does not form to any appreciable extent when the protein is expressed and folded in vivo in Escherichia coli.</p>

<p>In this project the aim has been to study the model protein carbonic anhydrase in Neisseria gonorrhoeae, a bacterium whose carbonic anhydrase has great similarities both structurally and functionally with the human form. By measuring and comparing the wild type of NGCA with mutants lacking one of the four tryptophan residues it can be seen what effect these tryptophans has on stability and activity and then compare with the known data of HCA II to learn more about their differences and similarities. The results from the stability and activity measurements are that the wild type is by far the most stable protein with W141L mutant coming thereafter.</p><p>From Trp-fluorescence and CO2-hydration measurement a clear two-transition steps (N→ I→ U) can be seen. This differs from earlier data where it instead only was a one-transition step for the wild type (N→U). The data is also very reliable and gives in most cases a perfect fit to the line. We also see this two-transition step for the other mutants stable enough, strengthening the theory further.</p><p>One fact that could be drawn from all the measurements is that when an intermediate is formed the ability for the enzyme NGCA to perform it’s catalytically ability is disabled.</p><p>Another thing is that the purification scheme of HCA II is not optimal to be directly applied to NGCA, despite the similarity in secondary and tertiary structure.</p>

<p>Fotosyntesen kallas den process som omvandlar ljusenergi till kemisk energi. Fotosyntesen sker i tylakoidmembranet och drivs av två stora proteinkomplex, fotosystem II (PSII) och fotosystem I (PSI) då de tillförs energi i form av fotoner. PSI-N är ett mindre protein på ca 10 kDa som ingår i PSI. På något sätt, som ännu inte är klarlagt, samverkar PSI-N med PSI-F och plastocyanin när det dockar till PSI. Det är därför av viktigt att rena fram större mängder av PSI-N för att få djupare kunskaper om proteinet samt dess struktur och funktioner. Tidigare undersökningar har utförts i ämnet och ett fusionsprotein innehållande PSI-N har uttryckts i <em>Escherichia coli</em> (<em>E.coli</em>). Problem har dock uppstått efter lysis av cellerna då det har visat sig att fusionsproteinet har degraderats. Vårt examensarbete strävar efter att rena fram intakt fusionsprotein med hjälp av, framför allt, mekanisk lysis och proteasinhibitorer.</p> / <p>The process where light is converted into chemical energy is called photosyntesis. The reaction takes place in the thylakoid membrane and is driven by two major protein complexes, photosystem II (PSII) and photosystem I (PSI) when energy in form of photons are received. PSI-N, a subunit in PSI, is a smaller protein with a mass of approximately 10 kDa. In some way, which is not yet clarified, PSI-N collaborates with PSI-F and plastocyanin when plastocyanin is docking to PSI. It is therefore important to purify larger amounts of the protein to acquire deeper knowledge of its structure and function. In earlier research the PSI-N protein has been expressed in <em>Escherichia coli</em> (<em>E.coli</em>). The problem has been degradation of the fusion protein after lysis. Our goal with this project is to obtain the purified protein intact using mechanic lysis and protease inhibitors.</p>

<p><!-- /* Font Definitions */ @font-face {font-family:Garamond; panose-1:2 2 4 4 3 3 1 1 8 3; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:647 0 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --></p><p>Several bacterial species are capable to use perchlorate and/or chlorate as an alternative electron acceptor in absence of oxygen. Microbial respiration of oxochlorates is important for biotreatment of effluent from industries where oxochlorates are produced or handled. One of these species, the Gram-negative <em>Ideonella dechloratans</em>, is able to reduce chlorate but not perchlorate. Two soluble enzymes, chlorate reductase and chlorite dismutase, participate in the conversion of chlorate into chloride and molecular oxygen. The present study deals with the electron transport from the membrane-bound components to the periplasmic chlorate reductase. Soluble <em>c</em> cytochromes were investigated for their ability to serve as electron donors to chlorate reductase. The results show that a 6 kDa <em>c </em>cytochrome serves as electron donor for chlorate reductase. This cytochrome also serves as electron donor for a terminal oxidase in the reduction of oxygen that is produced in the course of chlorate respiration. A gene encoding a soluble <em>c</em> cytochrome was found in close proximity to the gene cluster for chlorate reduction. This gene was cloned and expressed heterologously, and the resulting protein was investigated as a candidate electron donor for chlorate reductase. Electron transfer from this protein could not be demonstrated, suggesting that the gene product does not serve as immediate electron donor for chlorate reductase.</p><p> </p>

<p>alpha-actinin is a ubiquitous protein found in most eukaryotic organisms. The ability to form dimers allows alpha-actinin to cross-link actin in different structures. In muscle cells alpha-actinin is found at the Z-disk of sarcomeres. In non-muscle cells alpha-actinin is found in zonula adherens or focal adhesion sites where it can bind actin to the plasma membrane.</p><p>alpha-actinin is the shortest member of the spectrin superfamily of proteins which also includes spectrin, dystrophin and utrophin. Several hypotheses suggest that alpha-actinin is the ancestor of this superfamily.</p><p>The structure of alpha-actinin in higher organisms has been well characterized consisting of three main domains: an N-terminal actin-binding domain with two calponin homology domains, a central rod domain with four spectrin repeats and a C-terminal calcium-binding domain. Data mining of genomes from diverse organisms has made possible the discovery of new and atypical alpha-actinin isoforms that have not been characterized yet.</p><p>Invertebrates contain a single alpha-actinin isoform, whereas most of the vertebrates contain four. These four isoforms can be broadly classified in two groups, muscle isoforms and non-muscle isoforms. Muscle isoforms bind actin in a calcium independent manner whereas non-muscle isoforms bind actin in a calcium-dependent manner.</p><p>Some of the protozoa and fungi isoforms are atypical in that they contain fewer spectrin repeats in the rod domain. We have purified and characterized two ancestral alpha-actinins from the parasite Entamoeba histolytica. Our results show that despite the shorter rod domain they conserve the most important functions of modern alpha-actinin such as actin-bundling formation and calcium-binding regulation. Therefore it is suggested that they are genuine alpha-actinins.</p><p>The phylogenetic tree of alpha-actinin shows that the four different alpha-actinin isoforms appeared after the vertebrate-invertebrate split as a result of two rounds of genome duplication. The atypical alpha-actinin isoforms are placed as the most divergent isoforms suggesting that they are ancestral isoforms. We also propose that the most ancestral alpha-actinin contained a single repeat in its rod domain. After a first intragene duplication alpha-actinin with two spectrin repeats were created and a second intragene duplication gave rise to modern alpha-actinins with four spectrin repeats.</p>

<p>Nuts of all kinds are common ingredients in food. For nut allergy sufferers the frequent use of nuts cause problems and "hidden" nuts in food products may elicit allergic reaction when such foods are consumed. Methods for detecting and quantifying walnut (and other nuts) with high sensitivity and specificity are therefore very important.</p><p>The objective of this project was to verify the specificity of a rabbit antiserum against walnut with immunodiffusion and to determine the size of the dominant walnut antigens with Western blotting. In addition, a commercial sandwich ELISA for walnut quantification was validated and compared with a qualitative real-time PCR.</p><p>The rabbit antiserum proved to be less specific but after absorption with cross-reacting nuts and seeds it showed high specificity. The ELISA kit reacted, except for walnut, with pecan and slightly with other nuts and seeds tested. The PCR showed an absolute specificity to walnut. As low levels as 2.5mg walnut/kg can be quantified with the ELISA. This is 8 to 100 fold less than with the PCR method. It is therefore concluded that the ELISA kit is more sensitive than the PCR method but the PCR method is more specific than the ELISA kit.</p>

<p>Capillary electrophoresis (CE) is an analytical technique that is very useful for investigating processes that modify the charge and mass of proteins and polypeptide pharmaceuticals. This report explores the ability of CE to determine the aggregation constant between insulin molecules. Bovine insulin is a polypeptide (Mw=5733, pI = 5.3) that has two α-amino groups (Gly and Phe) and one ε–amino group (Lys). Analysis of concentration dependence of electrophoretic mobility of insulin at different conditions yields the association constant for dimerization of insulin. The association constant estimates how tight the peptide molecules are associated. The association constant is a useful factor to evaluate the purity of a peptide or protein sample.</p><p>The association reaction of bovine insulin molecules was found to be favoured by temperature. The association constants were 7200 M -1, 8000 M -1, and 36000 M -1 at 15 oC, 25 oC and 35 oC, respectively. The interactions between the peptide molecules increase at higher temperature, resulting in stronger association. The association constant was estimated to be 3000 M -1in the presence of dioxane (5%, w/v %) at 25 oC. However, the interaction sites remain to be explored.</p>

<p>The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term.</p><p>Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue.</p><p>The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta.</p><p>No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.</p>

<p>Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.</p><p>Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.</p><p>To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. </p><p>The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. </p><p>The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.</p>

<p>The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, <i>Hypocrea jecorina</i> and <i>Phanerochaete chrysosporium</i>, including both the action of the individual enzymes and their synergistic interplay. </p><p>The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases.</p><p>The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface.</p><p>A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases.</p><p>Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation.</p><p>Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.</p>