In this study, the aim was identification and classification of the enzyme having phenol oxidase activity produced by a thermophilic fungus, Scytalidium thermophilum. For this purpose, enzyme production, purification, biochemical characterization and structural analysis by X-ray crystallography studies have been performed. At the beginning of the research, this enzyme was considered as a phenol oxidase and analyzed accordingly. However, during purification, amino acid sequencing and structural studies, the enzyme was shown to be a catalase, with an additional catechol oxidase activity. This novel bifunctional catalase-catechol oxidase (CCO) was purified 10 fold with 45 % yield by anion exchange and gel filtration chromatographies. CCO was determined as a tetrameric protein having total and subunit molecular weights of 320 and 80 kDa, respectively. Isoelectric point of CCO was verified as 5.0. CCO catalase and catechol oxidase activities were characterized in terms of their kinetic behavior at different pH and temperatures. Depending on the substrate specificity and inhibitor studies of CCO, the phenol oxidase activity was determined as catechol oxidase but not tyrosinase or laccase. The best crystallization condition for CCO was determined and X-ray diffraction data was collected at the Daresbury Synchrotron Radiation Source (United Kingdom) at 2.7 &Aring / resolution. The preliminary structure was solved by molecular replacement method using Penicilium vitale catalase structure. CCO was verified to have a tetrameric structure with two homodimers and a metal center in each polypeptide chain.

<p>This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. </p><p>The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. </p><p>The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus.</p><p><b>Keywords:</b> protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein</p>

<p>This thesis concerns different areas of biomolecular nuclearmagnetic resonance spectroscopy (NMR). In the first part of thethesis a new formalism for simulations of NMR pulse sequencesis introduced. The formalism is derived both from classicalmechanics and quantum mechanics and is presented forhomonuclear and heteronuclear spin systems. The formalism hasalso been adapted to systems in chemical exchange. Simulationsof pulse sequences should be more straightforward using the newformalism.</p><p>In the second part of the thesis the NMR solution structuresof two ribosomal proteins are described. The ribosome isresponsible for protein production in all living cells and tounderstand the mechanism of the ribosome it is important toknow the three dimensional structure. In this thesis thestructures of S16 and S19, two of the proteins in the smallribosomal subunit, are presented. S16 is a mixed α /βprotein with a five-stranded parallel-antiparallel β-sheetand two α -helices. S19 is s mixed α/β proteinwith a three-stranded parallel-antiparallel β -sheet, oneα -helix and a short 3₁₀-helix.</p><p>In the third part of the thesis a program for semiautomaticassignment of NMR-spectra is presented. Assigning resonances inthe NMR spectrum is a labor-intensive process, which can takelong time. In semiautomatic assignment a computer program aidsthe user in finding assignments but leaves all decisions to theuser, thus speeding up the process. The program described inthis thesis is a new version of ANSIG, called Ansig forWindows. The program runs on PCs under Windows and has severaltools for semiautomatic assignment.</p><p><b>Keywords:</b>nuclear magnetic resonance, structuredetermination, ribosomal proteins, NMR simulations, NMR theory,NMR assignment software, semiautomatic assignment</p>

Electron crystallography is powerful for determination of complex structures. The newly-developed 3D electron diffraction (ED) methods make structure determination from nano- and micron-sized crystals much easier than using other methods, for example X-ray diffraction. Almost complete 3D ED data can be collected easily and fast from crystals at any arbitrary orientations. Dynamical effects are largely reduced compared to zonal ED patterns. 3D ED is powerful for phase identification and structure solution from individual nano- and micron-sized crystals, while powder X-ray diffraction (PXRD) provides information from all phases present in the samples. 3D ED methods and PXRD are complementary and their combinations are promising for studying multiphasic samples and complicated crystal structures. In this thesis, the feasibility and capability of 3D ED methods, specifically rotation electron diffraction (RED), in phase identification and structure determination of different kinds of crystalline materials with nano- or submicrometer-sized crystals are investigated. Experimental conditions for RED data collection and data processing in relation to data quality, as well as the challenges in the applications of RED are discussed. RED was combined with PXRD to identify phases from as-synthesized samples and to characterize atomic structures of eleven crystalline compounds. It was shown to be possible to identify as many as four distinct compounds within one sample containing submicron-sized crystals in a Ni-Se-O-Cl system. RED was also used to determine unit cell and symmetry of isoreticular metal-organic frameworks (SUMOF-7) and solve five zeolite structures with new frameworks, ITQ-51, ITQ-53, ITQ-54, EMM-23 and EMM-25 and that of a metal-organic framework (MOF), SUMOF-7I. The structure of an open-framework germanate SU-77 was solved by combining RED with PXRD. The structures of the zeolites and SU-77 were confirmed by Rietveld refinement against PXRD. High-resolution transmission electron microscopy was used to confirm the structure models of ITQ-51, EMM-25 and SUMOF-7I. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Accepted. Paper 6: Manuscript. Paper 7: Epub ahead of print. Paper 9: Manuscript. Paper 11: Manuscript.</p>

No description available.

A recent shift towards proteomics has seen many structural genomics initiatives set up for high-throughput structure determination using traditional methods of x-ray crystallography and NMR. The next step in the proteomic revolution focuses on the interplay of multi-protein complexes and transient protein-protein interactions, which are involved in many cellular functions. Greater understanding of protein-protein interactions will inevitably lead to better comprehension of the regulation of cellular process, which has implications in biomedical sciences and biotechnology. Even though many high-resolution initiatives focus on proteins and protein complexes, their structure-determination success rates are still low. An emerging approach uses chemical cross-linking and mass spectrometry to derive a set of sparse distance constrains, which can be used for building models of proteins and to map out residues in protein interaction interface based on partial structural information. This technique allows low-resolution identification of protein structures and their interactions in cases where traditional structure determination techniques did not produce results. Chemical cross-linkers have been successfully used for many years in identifying interacting proteins. However, recent advances in mass spectrometry have allowed the identification of exact insertion points of low-abundance cross-links and hence has opened up a new perspective on the use of cross-linkers in combination with computational structure prediction. For protein interaction studies, the approach uses chemical cross-linking information with molecular docking, so that the cross-links are treated as explicit constraints in the calculations. This study focuses on a low-cost and rapid approach to structure prediction, where partial structural information and distance constraints can be used to obtain the relative orientation of interacting proteins and domains, specifically as a rescue strategy where traditional high resolution structure determination methods were unsuccessful. This hybrid biochemical/bioinformatics approach was applied in the determination of structure of the latexin:carboxypeptidase A complex, and succeeded in achieving 4 Å rmsd compared to the crystal structure determined subsequently (Mouradov et al., 2006). Application of the bioinformatics/biochemical approach to multi-domain proteins was carried out on murine acyl-CoA thioesterase 7 (Acot7). X-ray crystallography provided structures of the two separate domains of Acot7, however the full length protein did not crystalise. Combining chemical cross-linking, mass spectrometry, molecular docking and homology modeling we were able to reconstruct how the two domains are arranged in the full length protein (Forwood et al., 2007). Limitations of this technique caused by the enormous complexity of the cross-linking reaction mixtures were identified and emphasized by analysing a large (four protein) complex of DNA polymerase III, where only one inter-protein cross-link was identified. A rapid and cost-effective method for identification of cross-linked peptides using a commercially available cross-linker was developed as part of the overall aim of streamlining the hybrid biochemical/bioinformatics in order for it to become a generally applicable technique for rapid protein structure characterisation (King et al., 2008). Finally an in-house software package was developed for assignment of cross-linked peptides based on m/z values.

A recent shift towards proteomics has seen many structural genomics initiatives set up for high-throughput structure determination using traditional methods of x-ray crystallography and NMR. The next step in the proteomic revolution focuses on the interplay of multi-protein complexes and transient protein-protein interactions, which are involved in many cellular functions. Greater understanding of protein-protein interactions will inevitably lead to better comprehension of the regulation of cellular process, which has implications in biomedical sciences and biotechnology. Even though many high-resolution initiatives focus on proteins and protein complexes, their structure-determination success rates are still low. An emerging approach uses chemical cross-linking and mass spectrometry to derive a set of sparse distance constrains, which can be used for building models of proteins and to map out residues in protein interaction interface based on partial structural information. This technique allows low-resolution identification of protein structures and their interactions in cases where traditional structure determination techniques did not produce results. Chemical cross-linkers have been successfully used for many years in identifying interacting proteins. However, recent advances in mass spectrometry have allowed the identification of exact insertion points of low-abundance cross-links and hence has opened up a new perspective on the use of cross-linkers in combination with computational structure prediction. For protein interaction studies, the approach uses chemical cross-linking information with molecular docking, so that the cross-links are treated as explicit constraints in the calculations. This study focuses on a low-cost and rapid approach to structure prediction, where partial structural information and distance constraints can be used to obtain the relative orientation of interacting proteins and domains, specifically as a rescue strategy where traditional high resolution structure determination methods were unsuccessful. This hybrid biochemical/bioinformatics approach was applied in the determination of structure of the latexin:carboxypeptidase A complex, and succeeded in achieving 4 Å rmsd compared to the crystal structure determined subsequently (Mouradov et al., 2006). Application of the bioinformatics/biochemical approach to multi-domain proteins was carried out on murine acyl-CoA thioesterase 7 (Acot7). X-ray crystallography provided structures of the two separate domains of Acot7, however the full length protein did not crystalise. Combining chemical cross-linking, mass spectrometry, molecular docking and homology modeling we were able to reconstruct how the two domains are arranged in the full length protein (Forwood et al., 2007). Limitations of this technique caused by the enormous complexity of the cross-linking reaction mixtures were identified and emphasized by analysing a large (four protein) complex of DNA polymerase III, where only one inter-protein cross-link was identified. A rapid and cost-effective method for identification of cross-linked peptides using a commercially available cross-linker was developed as part of the overall aim of streamlining the hybrid biochemical/bioinformatics in order for it to become a generally applicable technique for rapid protein structure characterisation (King et al., 2008). Finally an in-house software package was developed for assignment of cross-linked peptides based on m/z values.

abstract: CTB-MPR649-684 is a translational fusion protein consisting of the cholera toxin B subunit (CTB) and the conserved residues 649-684 of gp41 membrane proximal region (MPR). It is a candidate vaccine component aimed at early steps of the HIV-1 infection by blocking viral mucosal transmission. Bacterially produced CTB-MPR was previously shown to induce HIV-1 transcytosis-blocking antibodies in mice and rabbits. However, the induction of high-titer MPR specific antibodies with HIV-1 transcytosis blocking ability remains a challenge as the immuno-dominance of CTB overshadows the response to MPR. X-ray crystallography was used to investigate the structure of CTB-MPR with the goal of identifying potential solutions to improve the immune response of MPR. Various CTB-MPR variants were designed using different linkers connecting the two fusion proteins. The procedures for over-expression E. coli and purification have been optimized for each of the variants of CTB-MPR. The purity and oligomeric homogeneity of the fusion protein was demonstrated by electrophoresis, size-exclusion chromatography, dynamic light scattering, and immuno-blot analysis. Crystallization conditions for macroscopic and micro/nano-crystals have been established for the different variants of the fusion protein. Diffraction patterns were collected by using both conventional and serial femto-second crystallography techniques. The two crystallography techniques showed very interesting differences in both the crystal packing and unit cell dimensions of the same CTB-MPR construct. Although information has been gathered on CTB-MPR, the intact structure of fusion protein was not solved as the MPR region showed only weak electron density or was cleaved during crystallization of macroscopic crystals. The MPR region is present in micro/nano-crystals, but due to the severe limitation of the Free Electron Laser beamtime, only a partial data set was obtained and is insufficient for structure determination. However, the work of this thesis has established methods to purify large quantities of CTB-MPR and has established procedures to grow crystals for X-ray structure analysis. This has set the foundation for future structure determination experiments as well as immunization studies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

Orientador: Abner de Siervo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-16T08:29:21Z (GMT). No. of bitstreams: 1 Lussani_FernandoCesar1985-_M.pdf: 10633607 bytes, checksum: 81211aec04bbbd3665a1c673e003a714 (MD5) Previous issue date: 2010 / Resumo: Algumas evidências na literatura apontam para um possível ordenamento ferromagnético do Paládio. Na natureza, o Paládio é um metal de transição não ferromagnético. Filmes ultrafinos podem induzir ordenamento ferromagnético devido a diferenças nos parâmetros de rede entre o filme(Pd) e o substrato que o suporta. Neste trabalho, cresceram-se filmes ultrafinos de Paládio sobre o substrato de W(100). Para tanto, o substrato foi limpo por meio de tratamento térmico e bom- bardeamento de íons. Filmes de Paládio foram crescidos usando epitaxia por feixe atômico e obteve-se um filme com espessura de cinco monocamadas de Paládio. Esse filme foi caracterizado pelas técnicas de espectroscopia de fotoemissão e espalhamento de elétrons de baixa energia. Determinou-se a estrutura de superfície por meio da técnica de difração de fotoelétrons usando radiação síncroton (LNLS), oriunda da linha SXS, com energia do fóton de 1810 eV. Realizou-se o mesmo estudo para filmes de Paládio sobre substrato de Nb(100). Neste sistema, depositou-se Paládio sobre uma superfície reconstruída de NbO(3 x 1) - 2D. Caracterizou-se este filme e o arranjo estrutural foi obtido por meio da técnica de difração de fotoelétrons com fótons de raio-X AI Ka de energia 1486.6 eV. Em seguida calculamos, por meio da teoria do funcional da densidade, propriedades estruturais e eletrônicas do Páladio. A partir destes cálculos, os resultados encontrados na determinação estrutural foram analisados. Calculou-se a densidade de estados para alguns modelos de empacotamento e discutiu-se sobre a existência ou não de ordenamento ferromagnético nesses sistemas / Abstract: Some evidence in the literature suggest a possible ferromagnetic ordering of the Palladium. In nature, the Palladium is a non-ferromagnetic metal transition. Ultra- thin films can induce ferromagnetic ordering due to differences in lattice parameters between the film(Palladium) and the substrate that supports it. In this work, it has been grown Palladium ultrathin films on the substrate W(100). First, The substrate was cleaned by heat treatment and ion bombardment. Palladium films were grown using atomic beam epitaxy and it was obtained a film with a thickness of tive monolayers of Palladium. This film was characterized by X- ray photoelectron spectroscopy and by low energy electron diffraction. The surface structure has been determined by X-ray photoelectron diffraction technique using synchrotron radiation (LNLS), from SXS line with the photon energy of 1810 eV. We performed the same study for Palladium films over Nb(100). In this system, Palladium monolayers were deposited on a reconstructed surface of NbO(3 x 1)- 2D. These films has been characterized and the structural arrangement was achieved through the technique of photoelectron diffraction with photons from a AI Ka source with energy of 1486.6 eV. Then, it was calculated by density functional theory, the electronic and structural properties of Palladium. From these calculations, the results found in structural determination were analyzed. It has been calculated the density of states for some models of packaging and it was argued about the existence of ferromagnetic ordering in these systems / Mestrado / Física da Matéria Condensada / Mestre em Física

Electron crystallography is complementary to X-ray crystallography. Single crystal X-ray diffraction requires the size of a crystal to be larger than about 5 × 5 × 5 μm3 while a TEM allows a million times smaller crystals being studied. This advantage of electron crystallography has been used to solve new structures of small crystals. One method which has been used to collect electron diffraction data is rotation electron diffraction (RED) developed at Stockholm University. The RED method combines the goniometer tilt and beam tilt in a TEM to achieve 3D electron diffraction data. Using a high angle tilt sample holder, RED data can be collected to cover a tilt range of up to 140o.  Here the crystal structures of several different compounds have been determined using RED. The structure of needle-like crystals on the surface of NiMH particles was solved as La(OH)2. A structure model of metal-organic layers has been built based on RED data. A 3D MOF structure was solved from RED data. Two halide perovskite structures and two newly synthesized aluminophosphate structures were solved. For those beam sensitive crystals characterized here, sample cooling down to -170oC was used to reduce the beam damage. The low temperature not only reduces electron beam damage, but also keeps the structure more stable in the high vacuum in a TEM and improves the quality of the diffraction data. It is shown that cooling can improve the resolution of diffraction data for MOFs and zeolites, for samples undergoing phase changes at low temperature, the data quality could be worse by cooling. In summary, cooling can improve the ED data quality as long as the low temperature does not trigger structural changes. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Submitted.</p>