Bacterial L-asparaginases (E.C.3.5.1.1) are enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid. For the past 30 years these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. The presence of a low rate glutaminase activity however causes serious side-effects to patients in treatment, as glutamine depletion give rise to neurotoxicity, anaphylaxis, and other hypersensitivity reactions. The interest in the enzyme from Erwinia carotovora originates from the fact that it shows a decreased glutaminase activity, and therefore the enzyme is expected to exhibit fewer side effects when used in therapy. The main focus of this thesis is the crystal structure determination of L-asparaginase from Erwinia carotovora in the presence of aspartic acid at 2.5 Å resolution. The structure was refined to an R/Rfree factor of 19.9/28.6 with good stereochemistry. L-Asparaginases are homotetrameric enzymes with a known 222 symmetry and an identical fold. The Erwinia carotovora asparaginase consists of eight monomers of 330 amino acid residues each. In this case the enzyme is active as a dimer of tetramers. The two tetramers have an inner twofold non-crystallographic symmetry. Each monomer forms two identifiable domains a large N-domain and a small C-domain. The active sites are found at a topological switch-point between those domains.

The main topic of this thesis is the development of computational search methods that are useful in drug design applications. The emphasis is on exhaustiveness of the search method such that it can guarantee a certain level of geometric accuracy. In particular, the following two problems are addressed: (i) Prediction of binding mode of a drug molecule to a receptor and (ii) prediction of crystal structures of drug molecules. Predicting the binding mode(s) of a drug molecule to a target receptor is pivotal in structure-based rational drug design. In contrast to most approaches to solve this problem, the idea in this work is to analyze the search problem from a computational perspective. By building on top of an existing docking tool, new methods are proposed and relevant computational results are proven. These methods and results are applicable for other place-and-join frameworks as well. A fast approximation scheme for the docking of rigid fragments is described that guarantees certain geometric approximation factors. It is also demonstrated that this can be translated into an energy approximation for simple scoring functions. A polynomial time algorithm is developed for the matching phase of the docked rigid fragments. It is demonstrated that the generic matching problem is NP-hard. At the same time the optimality of the proposed algorithm is proven under certain scoring function conditions. The matching results are also applicable for some of the fragment-based de novo design methods. On the practical side, the proposed method is tested on 829 complexes from the PDB. The results show that the closest predicted pose to the native structure has the average RMS deviation of 1.06 °A. The prediction of crystal structures of small organic molecules has significantly improved over the last two decades. Most of the new developments, since the first blind test held in 1999, have occurred in the lattice energy estimation subproblem. In this work, a new efficient systematic search method that avoids random moves is proposed. It systematically searches through the space of possible crystal structures and conducts search space cuts based on statistics collected from the structural databases. It is demonstrated that the fast search method for rigid molecules can be extended to include flexible molecules as well. Also, the results of some prediction experiments are provided showing that in most cases the systematic search generates a structure with less than 1.0°A RMSD from the experimental crystal structure. The scoring function that has been developed for these experiments is described briefly. It is also demonstrated that with a more accurate lattice energy estimation function, better results can be achieved with the proposed robust search method.

<p>The recent development of methods for constructing andhandling large collections (libraries) of proteins, from whichvariants with desired traits can be isolated, hasrevolutionized the field of protein engineering. Key elementsof such methods are the various ways in which the genotypes(the genes) and the phenotypes (the encoded proteins) arephysically linked during the process. In one section of thework underlying this thesis, one such technique (phagedisplay), was used to isolateand identify protein librarymembers based on their catalytic or target molecule-bindingproperties.</p><p>In a first study, phage display libraries of the lipolyticenzyme Lipolase from Thermomyces lanuginosa were constructed,the objective being to identify variants with improvedcatalytic efficiency in the presence of detergents. Toconstruct the libraries, nine positions were targeted for codonrandomization, all of which are thought to be involved in theconformational change-dependent enzyme activation that occursat water-lipid interfaces. The aim was to introduce two tothree amino acid mutations at these positions per lipase gene.After confirming that the wt enzyme could be functionallydisplayed on phage, selections with the library were performedutilizing a mechanism-based biotinylated inhibitor in thepresence of a detergent formulation. According to rhodamineB-based activity assays, the fraction of active clonesincreased from 0.2 to 90 % over three rounds of selection.Although none of the variants selected using this approachshowed increased activity, in either the presence or absence ofdetergent compared to the wild type enzyme, the resultsdemonstrated the possibility of selecting variants of theenzyme based on catalytic activity.</p><p>In the following work, phage libraries of the StaphylococcalProtein A (SPA)-derived Z-domain, constructed by randomizationof 13 surface-located positions, were used to isolate Z domainvariants (affibodies) with novel binding specificities. Astargets for selections, the parental SPA domains as well as twopreviously selected affibodies directed against two unrelatedtarget proteins were used. Binders of all three targets wereisolated with affinities (KD) in the range of 2-0.5 µM.One SPA binding affibody (Z_SPA-1) was shown to bind to each of the fivehomologous native IgG-binding domains of SPA, as well as theZdomain used as the scaffold for library constructions.Furthermore, the Z_SPA-1affibody was shown to compete with one of thenative domains of SPA for binding to the Fc part of humanantibodies, suggesting that the Z_SPA-1affibody bound to the Fc-binding surface ofthe Z domain. The majority of the affibodies isolated in theother two selections using two different affibodies as targets,showed very little or no binding to unrelated affibodies,indicating that the binding was directed to the randomizedsurface of their respective targets, analogously toanti-idiotypic antibodies.</p><p>The structure of the wild type Z domain/Z_SPA-1affibody co-complex was determined by x-raycrystallography, which confirmed the earlier findings in thatthe affibody Z_SPA-1affibody was shown to bind to the Fc bindingsurface of the Z domain. Further, both the Z domain and the Z_SPA-1affibody had very similar three helix-bundletopologies, and the interaction surface involved ten out of thethirteen randomized residues, with a central hydrophobic patchsurrounded by polar residues. In addition, the interactionsurface showed a surprisingly high shape complementarity, giventhe limited size of the library used for selections. The Z_SPA-1affibody was further investigated for use invarious biotechnological applications. In one study, the Z_SPA-1affibody was successfully recruited as a novelaffinity gene fusion partner for production, purification anddetection of cDNA-encoded recombinant proteins using anSPA-based medium for affinity chromatography. Further, the SPAbinding capability of the Z_SPA-1affibody was employed for site-specific andreversible docking of Z_SPA-1affibody-tagged reporter proteins onto an SPAfusion protein anchored to a cellulose surface via acellulose-binding moiety. These generated protein complexesresembles the architecture of so-called cellulosomes observedin cellulolytic bacteria. The results suggest it may bepossible to use anti-idiotypic affibody-binding protein pairsas modules to build other self-assembling types of proteinnetworks.</p><p><b>Keywords:</b>phage display, selection, mechanism-basedinhibitor, affinity domains, crystal structure, Staphylococcusaureus protein A, affinity chromatography, anti-idiotypicbinding pairs, affibody, combinatorial, protein engineering,lipase, cellulosome, assembly.</p>

Cavitands are very important class of compounds in supramolecular chemistry. These molecules contain rigid enforced cavity,and have attracted considerable attention in supramolecular chemistry as building blocks for the construction of carcerands, hemicarcerands, and other host guests complexes. Nearly 40 years ago, Niederl and Vogel laid foundation for the study of such type of condensation reactions. In our laboratory we are involved in synthesis of resorcinarenes with readily available substrates such as resorcinol and aldehydes to form a cyclic tetramer. Herein, I present detailed studies about the functionalization of the synthesized tetramers and their antimicrobial activity. Octahydroxy resorcinarenes were synthesized and perallylated which served as acyclic diene precursors for ring closing metathesis reaction. Studies were carried out to see effect of C-2 substituent of resorcinol and effect of aryl substituents, and aliphatic substituents on ring closing metathesis. This thesis describes the synthesis of bridged resorcinarenes and study of antimicrobial activity of resorcinarenes.

Intramolecular ring closing metathesis in the presence of Grubbs' catalyst has been used as an efficient approach to synthesize bridged resorcinarenes. Octaallyl cavitands may undergo conformational changes; however bridge formation by RCM of the allyl groups gives a rigid, enforced, concave cavity capable of holding neutral molecules. This is the first report describing tandem formation of the four bridges on the upper rim of resorcinarenes. Structures of bridged resorcinarenes are confirmed by spectral analysis data.This report also describes the synthesis of polyhydroxy resorcinarenes, which have been used as metal complexing agents, sensors, receptors, molecular reaction vessels and catalytic chambers. They are able to encapsulate small neutral molecules, drug molecules inside the cavity. Such cavitands offer unique molecular platforms for host--guest chemistries, as well as new polymers and self-assembled systems.

The availability of large amounts of data generated by high-throughput computing and experimentation has generated interest in the application of machine learning techniques to materials science. Machine learning of materials behavior requires the use of feature vectors that capture compositional or structural information influence a target property. We present methods for assessing the similarity of compositions, substructures, and crystal structures. Similarity measures are important for the classification and clustering of data points, allowing for the organization of data and the prediction of materials properties. / Engineering and Applied Sciences

Lipoxygenases (LOX) catalyze regio- and stereospecific oxygenation of polyunsaturated fatty acids to hydroperoxides. These hydroperoxides are further metabolized to leukotrienes and lipoxins in mammals, and are involved in asthma and inflammation. LOX of animals and plants contain iron as catalytic metal (FeLOX). Filamentous fungi use both FeLOX, and manganese containing LOX (MnLOX). The role of LOX in fungi is still not known. This thesis focuses on expression of novel MnLOX, analyses of their reaction mechanism and products by HPLC-MS/MS, protein crystallization and analysis of the first MnLOX structure.   MnLOX from G. graminis, M. salvinii, M. oryzae, F. oxysporum and C. gloeosporioides were expressed in Pichia pastoris, purified and characterized by HPLC-MS/MS. All MnLOX catalyzes suprafacial hydrogen abstraction and oxygen insertion. Replacement of one Ile to Phe in the active site of MnLOX of G. graminis could switch the mechanism from suprafacial to mainly antarafacial. MnLOX of F. oxysporum was interesting since it catalyzes oxygenation of linoleic acid to 11R- instead of the more common 11S-hydroperoxides. This feature could be attributed to a single Ser/Phe exchange in the active site.   We found that Gg-MnLOX utilizes hydrogen tunneling in the reaction mechanism, but was slightly more temperature dependent than soybean FeLOX. It is an intriguing question why some fungal LOX use manganese and not iron as catalytic metal and whether the large redox potential of Mn2+/Mn3+ (1.5 V) can be tuned close to that of Fe2+/Fe3+ (0.77 V) for redox cycling and catalysis. We present crystallization conditions for two MnLOX, and the 2.07 Å crystal structure of MnLOX from M. oryzae, solved using sulfur and manganese single anomalous dispersion (SAD). The structure reveals a similar metal coordinating sphere as FeLOX but the metal ligand Asn473 was positioned on a short loop instead of a helix and formed interactions with a conserved Gln. This feature could be essential for the use of manganese as catalytic metal in LOX. We found three Phe residues that likely facilitate the suprafacial hydrogen abstraction and oxygen insertion for MnLOX. These findings provide new insight into the unique reaction mechanism of MnLOX.

The main causative agent of human urinary tract infections is the uropathogenic Escherichia coli (UPEC) pathotype. It may cause disease due to its ability to express a number of bacterial virulence factors. Fimbrial adhesins are particularly important for the initial establishment of infection in the urinary tract. The fimbriae are hair-like structures protruding from the bacterial cell and by attaching to specific receptors in the urinary tract they mediate adherence to different cell types, allowing the bacteria to resist the shear forces from urine flow. The UPEC strains generally carry multiple determinants for fimbrial adhesins. Previous studies have indicated that there is a co-regulation between different fimbrial genes and one factor that has been implicated in this is the PapB protein, acting as a transcriptional regulator of P-fimbrial expression. The PapB protein can be regarded as the prototype of a family of fimbrial regulators that show high homology between different fimbrial operons. One homolog is FocB, regulator of F1C fimbriae. In this study, the role of the FocB protein in the regulation of F1C fimbriae as well as in the co-regulation with other fimbrial genes was investigated. It was observed that FocB binds to DNA, similarly to PapB, in an oligomeric fashion and that PapB and FocB can form hetero-oligomeric complexes, which appear to have a repressive role in the regulation of the F1C fimbriae. In addition, the FocB protein also had a repressive effect on transcription of the fim operon, which encodes theType 1 fimbriae. For further analysis of FocB in vitro, we developed efficient procedures for purification of the protein and established conditions for its crystal formation with the aim to conduct X-ray diffraction studies. By the hanging-drop vapour-diffusion method, we obtained crystals that in the X-ray analysis diffracted sufficiently well to allow modelling of a high resolution structure of FocB. The structural model was considered in relation to the DNA binding properties of the protein. The FocB analysis represents the first structural model of this family of transcriptional factors. This model should aid in further understanding of the roles and functions of these proteins in the regulation of the UPEC fimbrial operons. The complexity of the system, with multiple factors involved in the regulation of fimbrial operons, was revealed in earlier studies of the PapI protein showing that PapI activates transcription of the pap operon as a part of a complex with the global regulator Lrp. However, PapI itself did not appear to bind to DNA and its mode of action has remained unclear. By genetic analyses and in vitro studies we show that PapI may interact also with the α subunit of the RNA polymerase. This finding indicates that PapI might directly interact with the transcriptional apparatus and thus aid in the activation of pap expression. Bacteria are frequently releasing outer membrane vesicles (OMVs) from their surface. We studied the release of the haemolysin toxin from E. coli in connection with formation of OMVs and found that the toxin was tightly associated with the vesicles in an active form. By overproduction of the PapB or PapI regulators in order to maximise the population of bacteria expressing fimbriae, we could detect P fimbriae proteins associated with OMVs that displayed specific adhesion to receptor-coated beads. This suggests a possible scenario in which the vesicles canfunction as directed vehicles of bacterial virulence factors.

Enormous amount of latent heat generates/absorbs at the formation/dissociation process of gas hydrates and controlls their thermal condition themselves. In this paper we investigated the effect of ethane concentration on dissociation heat of mixed-gas (methane and ethane) hydrate. It has been reported by researchers that a structure II gas hydrate appears in appropriate gas composition of methane and ethane. We confirmed by using Raman spectroscopy that our samples had the following three patterns: structure I only, structure II only and mixture of structures I and II. Dissociation heats of the mixed-gas hydrates were within the range between those of pure methane and ethane hydrates and increased with ethane concentration. In most cases two peaks of heat flow appeared and the dissociation process was divided into two parts. This can be understood in the following explanation that (1) the sample contained both crystal structures, and/or (2) ethane-rich gas hydrate formed simultaneously from dissociated gas and showed the second peak of heat flow.

II tipo restrikcijos endonukleazės atpažįsta specifines DNR sekas ir kerpa DNR šiose sekose arba šalia jų. BpuJI, atpažįstanti 5’-CCCGT seką, skiriasi nuo kitų fermentų tuo, kad jos kirpimo vieta yra labai variabili. Čia parodoma, kad BpuJI yra dimeras, sudarytas iš dviejų monomerų, kurie turi po du atskirus domenus. BpuJI N domenas atpažįsta taikinį kaip monomeras, o C-domenas pasižymi nukleaziniu aktyvumu ir dimerizuojasi. Apo-fermento nukleazinis aktyvumas yra nuslopintas. N-domenams atpažinus taikinį, aktyvuojamas C-domenas, kuris perkerpa DNR šalia taikinio. Be to, aktyvuotas C-domenas yra nespecifinė nukleazė, linkusi nukirpti ~3 nt nuo buko dvigrandės DNR galo. Taigi, BpuJI DNR karpymo pobūdis yra labai sudėtingas. Bioinformatinė analizė ir kryptinga mutagenezė parodė, kad BpuJI C-domenas turi PD-(D/E)XK struktūrinę sanklodą ir yra panašus į archėjų Holidėjaus jungtis karpančias nukleazes. Išsprendus 1,3 Å skiriamosios gebos BpuJI N-domeno/DNR komplekso erdvinė struktūrą, paaiškėjo, kad šį domeną sudaro du „sparnuotą“ spiralė-linkis-spiralė motyvą turintys subdomenai. BpuJI taikinį atpažįsta aminorūgštys, esančios N-rankoje ir abiejų spiralė-linkis-spiralė motyvų atpažinimo spiralėse. BpuJI N-domenas yra labiausiai panašus į Nt.BspD6I nukleazę, kerpančią vieną DNR grandinę. Nt.BspD6I/DNR komplekso struktūros modelis rodo, kad Nt.BspD6I ir BpuJI taikinį atpažįstantys struktūriniai elementai yra panašūs. / Type II restriction endonucleases recognize specific DNA sequences and cleave DNA at fixed positions within or close to this sequence. BpuJI recognizes the 5’-CCCGT sequence, but in contrast to other enzymes its cleavage site is very variable. This study shows that BpuJI is a dimer in solution and consists of two separate domains. The N-domain binds to the target sequence as a monomer, while the C-domain is responsible for nuclease activity and dimerization. The nuclease activity is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the N-domains. The activated C-domain cleaves DNA near the target site. In addition, it possesses an end-directed nuclease activity and preferentially cuts ~3 nt from the 3’ terminus. This leads to a very complicated pattern of DNA cleavage. Bioinformatics and mutational analysis revealed that the BpuJI C-domain harbours a PD (D/E)XK active site and is structurally related to archaeal Holliday junction resolvases. The crystal structure of the BpuJI N-domain bound to cognate DNA was solved at 1.3 Å resolution. It revealed two winged-helix subdomains, D1 and D2. The recognition of the target sequence is achieved the amino acid residues located on both the HTH motifs and an N-terminal arm. The BpuJI DNA recognition domain is most similar to the nicking endonuclease Nt.BspD6I. The modelling suggests that Nt.BspD6I could share the specificity-determining regions with BpuJI.