Crk associated substrate (Cas) is an adaptor protein that becomes phosphorylated upon integrin signaling and influences regulation of cell processes such as migration, proliferation and survival. It consists of multiple domains and regions that can interact with several signaling proteins involved in different signaling pathways. Cas was first discovered as a highly phosphorylated protein in v-Src and v-Crk transformed cells, showing involvement of this protein in cell transformation High level of Breast cancer antiestrogen resistance protein (BCAR-1), a homologue to Cas has shown to correlate with rapid reoccurrence of breast cancer and also create resistance towards Tamoxifen, the widely used medicine for receptor positive breast cancer patients. We have defined boundaries of two regions of Cas termed serine rich region (SRR) and Src binding domain (SBD) respectively and have isolated these segments for biochemical and structural studies. The structure of the serine rich part of Cas has been determined by NMR spectroscopy and reveals a four-helix bundle with unusually long loops. The 14-3-3 protein binds to Cas in a phospho-serine dependent manner and our study suggests that the binding site is located between two helices. The SH2-SH3 domain of a Src family kinase, Lck has also been crystallized in complex with a nine residue long peptide corresponding to the region in Cas that binds to SH2 domains. The structure of this complex has been solved at 2.7Å and shows that Cas binds Src family kinases (SFK) with high affinity suggesting a specific interaction between these two molecules. The biochemical studies on the specific binding site of these molecules show that SFK can bind to any of the phosphorylated tyrosines on the SH2 binding domain of Cas and only one phospho-tyrosine is enough to establish the binding. This binding assay does also indicate that SH3 binding domain of Cas is not essential for SFK binding.

Mucin is a term used to describe a large family of heavily glycosylated proteins which are present on the surfaces of secretory epithelial cells and are overexpressed by many carcinomas. Membrane-bound mucin MUC1 is of special interest. Its backbone consists of repeating units of twenty amino acids with five potential glycosylation sites. These sites are expanded to structures like the T (Galβ(1->3)GalNAcα-Ser/Thr) and Tn (GalNAcα-Ser/Thr) antigens by the action of various glycosyltransferases. In different types of carcinomas these epitopes are being terminated by sialic acid residues to form among others: 2,3-sialyl-T and sialyl-Tn structures due to the elevated levels of different sialyltransferases. Solid-phase synthesis of the selected antigens derived from the mucin MUC1 has been developed and optimized. A chemoenzymatic approach has been used to effectively prepare 2,3-sialyl-T and 2,6-sialyl-Tn glycopeptides. The formation of intramolecular sialic acid lactones in presence of acetic acid was investigated. The stability of lactones formed from 2,3-sialyl-T towards water was studied using NMR spectroscopy and it appeared that 1''->2' lactone displayed remarkable strength to hydrolysis and it was suggested as a candidate for cancer vaccine. Gel-phase 19F NMR spectroscopy is known to be a very good tool to characterize resin-bound products using fluorinated protecting groups and linker molecules. The hydrophobic peptide LLLLTVLTV, which is a fragment from the MUC1 signal sequence, was prepared using solid-phase synthesis according to a modified Fmoc protocol with more active coupling reagent, stronger base, and the isopropylidene dipeptide Fmoc-Leu-Thr-(ΨMe,Mepro)-OH. Gel-phase 19F NMR spectroscopy was used to evaluate peptide chain aggregation and coupling and deprotection efficiency. A carbamate linker strategy proved to be effective in solid-phase synthesis of serine-based neoglycolipids with terminal amino functionality. Neoglycolipids were covalently bound to secondary amines in microtiter plates using squaric acid ester methodology. These arrays have potential to study the interactions between carbohydrates and e.g. proteins and microbes. The new fluorinated α-amino protective group [1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] Fde was developed. This group is cleaved with hydrazine in DMF solution. By using amino acids protected with this group, it was possible to quantify the efficiency of peptide coupling using gel-phase 19F NMR spectroscopy.

Brominated organic compounds (BOCs) of both natural and anthropogenic origin are abundant in the environment. Most compounds are either clearly natural or clearly anthropogenic but some are of either mixed or uncertain origin. This thesis aims to identify some naturally produced BOCs and to develop a method for analysis of the bromine isotopic composition in BOCs found in the environment. Polybrominated dibenzo-p-dioxins (PBDDs) in the Baltic Sea are believed to be of natural origin although their source is unknown. Since marine sponges are major producers of brominated natural products in tropical waters, BOCs were quantified in a sponge (Ephydatia fluviatilis) from the Baltic Sea (Paper I). The results showed that the sponge does not seem to be a major producer of PBDDs in the Baltic Sea. In this study, mixed brominated/chlorinated dibenzo-p-dioxins were however discovered for the first time in a background environment without an apparent anthropogenic source. The use of nuclear magnetic resonance spectroscopy (NMR) is unusual in analytical environmental chemistry due to its sample requirements. Preparative capillary gas chromatography was used to isolate a sufficient amount of an unidentified BOC from northern bottlenose whale (Hyperoodon ampullatus) blubber (Paper II) to enable NMR analysis for identification of the compound. The bromine isotopic composition of BOCs may give information on the origin and environmental fate of these compounds. The first steps in this process are the development of a method to determine the bromine isotope ratio in environmentally relevant BOCs (Paper III) and measuring the bromine isotope ratio of several standard substances to establish an anthropogenic endpoint (Paper IV). / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details. This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques. The in vivo function and modularity of the membrane-anchored cellulase was also addressed using overexpression and complementation analysis in Arabidopsis thaliana. Among 9 genes found in the Populus EST database, encoding enzymes from glycosyl hydrolase family 9, two were expressed in the cambial tissue, and the membrane-anchored cellulase, PttCel9A1, was the most abundant transcript. PttCel9A1 was expressed in Pichia pastoris, and purified by affinity chromatography and ion exchange chromatography. The low yield of recombinant protein from shake flask experiments was improved by scaling up in the fermentor. PttCel9A1 was however highly heterogenous, both mannosylated and phosphorylated, which made the protein unsuitable for crystallization experiments and 3D X-ray structure determination. Instead, a homology model using a well-characterized, homologous bacterial enzyme was built. From the homology model, interesting point mutations in the active site cleft that would highlight the functional differences of the two proteins could be identified. The real-time cleavage patterns of cello-oligosaccharides by mutant bacterial enzymes, the wildtype bacterial enzyme and PttCel9A1 were studied by 1H NMR spectroscopy, and compared with results from HPAEC-PAD analysis. The inverting stereochemistry for the hydrolysis reaction of the membrane-anchored poplar cellulase was also determined by 1H NMR spectroscopy, and it was concluded that transglycosylation in vivo is not a possible scenario. The preferred in vitro polymeric substrates for PttCel9A1 were shown to be long, low-substituted cellulose derivatives, and the endo-1,4--glucanase activity was not extended to branched or mixed linkage substrates to detectable levels. This result indicates an in vivo function in the hydrolysis of “amorphous” regions of cellulose, either during polymerization or crystallization of cellulose. In addition, overexpressing PttCel9A1 in A. thaliana, demonstrated a correlation with decreased crystallinity of cellulose. The significance of the different putative modules of PttCel9A1 was investigated by the construction of hybrid proteins, that were introduced into a knock-out mutant of A. thaliana, and the potential complementation of the phenotype was examined. A type B plant cellulase catalytic domain could not substitute for a type A plant cellulase catalytic domain, although localization and interaction motifs were added to the N- and C-terminus. / QC 20100802

The thesis describes the realization of an asymmetric [2,3]-sigmatropic rearrangement of achiral allylic amines. It is divided into two parts; the first part deals with the development of a Lewis acid-mediated [2,3]-sigmatropic rearrangement and the second the asymmetric version thereof. Quaternization of an -amino amide with various Lewis acids established BBr3 and BF3 to be the most appropriate ones. Various allylic amines were subsequently rearranged into the corresponding homoallylic amines in good to excellent syn-diastereoselectivities, revealing the endo-transition state to be the preferred pathway. The structures of the intermediate Lewis acid-amine complexes were confirmed by NMR spectroscopy studies and DFT calculations. Based on this investigation a chiral diazaborolidine was chosen as Lewis acid and was shown to efficiently promote the asymmetric [2,3]-sigmatropic rearrangement furnishing homoallylic amines in good yields and excellent enantiomeric excesses. In contrast to the achiral rearrangement mediated by BBr3 and BF3, the asymmetric version gave the opposite major diastereomer, revealing a preference for the exo-transition state in the asymmetric rearrangement. To account for the observed selectivities, a kinetic and thermodynamic pathway was presented. On the basis of a deuterium exchange experiment on a rearranged Lewis acid-amine complex and an NMR spectroscopic investigation, the kinetic pathway was shown to be favored. / QC 20100927

Cancer is the second leading cause of death in the United States. Cisplatin is one of the well-known anti-cancer agents used to treat testicular and ovarian cancers. It mainly binds to the DNA bases, which leads to cell death. The cytotoxic activity of the cisplatin analogs is due to the interaction of platinum with nucleotides like adenine at N7 or N1 position and guanine at N7 position. Guanine is the primary target for cisplatin analogs whereas adenine is the secondary target. Cisplatin analogs, [Pt(Me5dien)(D2O)]2+[Me5dien = N,N,N’,N’,N’’-pentamethyl diethylene triamine] and [Pt(dien)(D2O)]2+[dien=diethylene triamine] were synthesized and their effects on AMP and GMP were studied using NMR spectroscopy. The experiments were conducted to examine the effects of bulk on 5’-GMP and 5’-AMP. The results suggest that bulk slows down the reaction with AMP more than with that of GMP. The order of reactivity is Pt(dien)(GMP)> Pt(dien)(AMP) > Pt(Me5dien)(GMP) > Pt(Me5dien)(AMP). The reaction of the [Pt(Me5dien)(D2O)]2+ complex with AMP leads to multiple products, some of which appear to be due to coordination at N1 instead of N7.

The research described in this thesis contributes to the development of new strategies facilitating advanced understanding of the fundamental principles governing selective recognition of molecularly imprinted polymers (MIPs) at a molecular level for the rational optimization of biomimetic materials. The nature of non-covalent interactions involved in the templating process of molecularly imprinted polymers based on the self-assembly approach were investigated with a variety of analytical techniques addressing molecular level interactions. For this purpose, the concerted application of IR and 1H-NMR spectroscopy enabled studying the complexation of the template molecules 2,4-dichlorophenoxyacetic acid, quercetin, and o-, m-, and p-nitrophenol with a variety of functional monomers in the pre-polymerization solution by systematically varying the ratio of the involved components. In aqueous and non protic porogenic solvents, information on the interaction types, thermodynamics, and complex stoichiometry was applied toward predicting the optimum imprinting building blocks and ratios. Molecular dynamics simulations of 2,4-dichlorophenoxyacetic acid and its interactions with the functional monomer 4-vinylpyridine in aqueous and aprotic explicit solvent allowed demonstrating the fundamental potential of computer MD simulations for predicting optimized pre-polymerization ratios and the involved interaction types. The obtained results clearly demonstrate that the application of rapid IR/NMR pre-screening methods in combination with molecular modeling strategies is a promising strategy towards optimized imprinting protocols in lieu of the conventionally applied labor intensive and time-consuming trial-and-error approach. Furthermore, HPLC characterization of the produced MIPs compared to control polymers enabled a systematic approach to imprinting based on advanced understanding of the factors governing the formation of high-affinity binding sites during the polymerization. In addition, the importance of the combination of size, shape, and molecular functionalities for the selective recognition properties of MIPs was investigated. MIPs for the mycotoxins deoxynivalenol and zearalenone and for the antioxidant quercetin were applied as separation materials for advanced sample preparation in beverage analysis. The obtained results demonstrated the potential of MIPs for rapid one-step sample clean-up and pre-concentration from beverages such as wine and beer.

<p>Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the Z_SPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: Z_Taq, originally selected to bind Taq DNA polymerase, and anti-Z_Taq, an anti-idiotypic binder to Z_Taq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free Z_SPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Ų total surface area and 10 out of 13 varied residues in Z_SPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of Z_SPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free Z_SPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of Z_SPA-1 upon binding seriously reduces the binding affinity. The Z_Taq:anti-Z_Taq complex buries in total 1672 Ų surface area and all varied positions in anti-Z_Taq are directly involved in binding. The main differences between the Z:Z_SPA-1 and the Z_Taq:anti-Z_Taq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither Z_Taq nor anti-Z_Taq shows the molten globule behaviour seen for Z_SPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.</p>

Das Zytokin Interleukin-10 (IL-10) ist ein Schlüsselspieler in der Regulation des Immunsystems mit pro- und anti-inflammatorischen Funktionen. Es spielt eine wichtige Rolle bei der Terminierung und Unterdrückung einer Entzündungsantwort, die ansonsten zu einer bleibenden Schädigung des Gewebes führen kann. Eine Dysregulation von IL-10 ist mit verschiedenen Krankheitsbildern wie chronischen Entzündungen, Autoimmunerkrankungen und Krebs assoziiert. IL-10 wird von einem breiten Spektrum von Zelltypen, darunter hauptsächlich hämatopoetische Zellen, aber auch epitheliale und mesenchymale Zellen, gebildet und in den extrazellulären Raum freigesetzt, wo es mit Komponenten der extrazellulären Matrix in Kontakt kommt. Es ist bekannt, dass IL-10 an Glykosaminoglykane (GAGs) binden kann und dass diese Interaktion seine biologische Aktivität beeinflusst. GAGs sind eine Klasse linearer Polysaccharide der extrazellulären Matrix. Sie bestehen aus wiederholenden Disaccharideinheiten und haben einen hoch negativ geladenen Charakter, welcher durch einen hohen Grad an Sulfatierung in der Zuckerkette zustandekommt. Sie binden eine Vielzahl an Signalproteinen und regulieren deren biologische Funktionen, etwa indem sie Einfluss auf die Rezeptorbindung oder die räumliche Verteilung des Proteins im Gewebe nehmen. Die molekularen Mechanismen, wodurch GAGs die biologische Aktivität von IL-10 beeinflussen, sind bisher unbekannt. Insbesondere ist nichts über die strukturellen Grundlagen der Interaktion bekannt, die Voraussetzung für ihr funktionelles Verständnis sind. In dieser Arbeit wurden daher die Bindungseigenschaften von IL-10 und GAGs sowie der strukturelle Aufbau ihres molekularen Komplexes unter Verwendung von NMR-Spektroskopie in Lösung charakterisiert. Es wurde eine definierte GAG-Bindungsstelle in IL-10 identifiziert und die Bindungsepitope und Bindungsaffinitäten von GAGs bestimmt. Die Ergebnisse dieser Arbeit weisen auf eine wichtige Rolle, die GAGs in der Biologie von IL-10 spielen können, hin – etwa für seine Speicherung im Gewebe oder für die IL-10-Rezeptorbindung.

In our society in the 21st century one of the greatest challenges is to provide raw materials to the industry in a sustainable way. This requires increased use of renewable raw materials such as wood. Wood is widely used in pulp, paper and textile industries and ongoing research efforts aim to extend the use of wood in e.g. liquid biofuels and green chemicals. At Umeå Plant Science Center (UPSC) poplar trees are used as model systems to study wood formation. The objective is to understand the function of the genes underlying the wood forming process. This knowledge could result in improved chemical and physical wood properties suitable for different industrial processes. This will in turn meet the demands for a sustainable development. This thesis presents tools and strategies to unravel information regarding genetic perturbations in poplar trees by the use of nuclear magnetic resonance (NMR) spectroscopy and multivariate analysis (MVA). Furthermore, gas chromatography/mass spectrometry (GC/MS) is briefly discussed in this context. Multivariate methods to find patterns and trends in NMR data have been used for more than 30 years. In the beginning MVA was applied in pattern recognition studies in order to characterize chemical structures with different ligands and in different solvents. Today, the multivariate methods have developed and the research have changed focus towards the study of biofluids from plant extracts, urine, blood plasma, saliva etc. NMR spectra of biofluids can contain thousands of resonances, originating from hundreds of different compounds. This type of complex data can be hard to summarize and interpret without appropriate tools and require sophisticated strategies for data evaluation. Related fields of research are rapidly growing and are here referred to as metabolomics. Five different research projects are presented which includes analysis of poplar samples where macromolecules such as pectin and also small molecules such as metabolites were analysed by high resolution magic angle spinning (HR/MAS) NMR spectroscopy, 1H-13C HSQC NMR and GC/MS. The discussion topics include modelling of metabolomic time dependencies in combination with genetic variation, validation of orthogonal projections to latent structures (OPLS) models, selection of putative biomarkers related to genetic modification from OPLS-discriminant analysis (DA) models, measuring one of the main components found in the primary cell-walls of poplar i.e. pectin, the use of Fourier transformed two-dimensional (2D) NMR data in OPLS modelling and model complexity in a PLS model.