Phosphorylated molecules play vastly important roles in the environment and biological systems. The main focus of this work sought to expand the current collection of synthetic phosphate receptors to include the binding of oligosaccharide phosphates. To this end, the synthesis of a boronic acid - appended porphyrin whose selectivity could be tuned through nucleic acid selection was pursued through a number of synthetic routes. Though difficulties were encountered with synthetic reactivities and product solubility, these efforts culminated in the development of a bis-boronic acid-substituted porphyrin soluble in DMSO/water mixtures that displays fluorescence quenching upon the addition of specific saccharide derivatives. In efforts to ease the synthetic burden, the creation of a DNA-based self-assembled receptor system was also investigated. Further, this work included the synthesis of phosphorylated derivatives of gemcitabine, a nucleoside chemotherapeutic drug, with the goal of developing targeted delivery systems for the improved treatment of cancer. Progress in each of these areas is discussed. / text

Binding of inorganic phosphate (Pi) in ATP synthase catalytic sites is a crucial step for the synthesis of adenosine-5'-triphosphate (ATP). ATP is the fundamental means of cellular energy in almost every organism, and in order to gain insight into the regulation of ATP catalysis, critical amino acid residues responsible for binding Pi must be identified. Here, we investigate the role of highly conserved α-subunit VISIT-DG sequence residues αSer-347, αGly-351, and αThr-349 in Pi binding. Mutations αS347A/Q, αG351Q, αT349A/D/R, βR182A, and αT349R/βR182A were generated via site directed mutagenesis. Results from biochemical assays showed that αSer-347 is required for transition state stabilization and Pi binding whereas αGly-351 is only indirectly involved in Pi binding and most likely maintains structural integrity of the catalytic site. Results from preliminary experiments on αThr-349 mutants suggest that the residue may be involved in Pi binding; however, further investigation is required to fully test this hypothesis.

Bakgrund: Fosfatbindande läkemedel är en viktig del i hemodialyspatienters behandling. Läkemedlet hjälper till att avlägsna fosfat ifrån kroppen, eftersom hyperfosfatemi annars ökar risken för kardiovaskulära sjukdomar. Trots den kunskapen har tidigare forskning visat att den kunskapen ibland uteblir hos hemodialyspatienter. Syfte: Syftet med studien var att undersöka hemodialyspatienters upplevelse av kunskap angående fosfatbindande läkemedel. Metod: En kvantitativ enkätundersökning på en hemodialysenhet i Mellansverige. Där svaren redovisas med deskriptiv statistik. Resultat: Studien visar på brister i hemodialyspatienters upplevelse av kunskap angående fosfatmedicinering både vad det gäller bristande information från sjukvårdspersonal men också brist på motivation att ta reda på informationen på egen hand. Dessutom visar studien på brister vad det gäller hemodialyspatienters förståelse till vikten av fosfatmedicinering. Slutsats: Studien visar att cirka hälften av hemodialyspatienterna upplever att de har kunskap för att kunna administrera fosfatbindande läkemedel på ett korrekt sätt. Men det finns fortfarande brister hos en del patienter vad det gäller kunskapen om fosfatbindande läkemedel. / Background: Phosphate-binding drugs are an important part of hemodialysis patient´s treatment because the drug helps to remove phosphate from there body, which otherwise increases the risk of cardiovascular disease. Previous research has shown that this knowledge is sometimes missing by hemodialysis patients. Objective: The aim of the study was to investigate the experience of knowledge about phosphate-binding drugs by hemodialysis patients. Method: A quantitative surveys at a hemodialysis unit in the central of Sweden. Where the answers are reported with descriptive statistics. Results: The study shows deficiencies in hemodialysis patients’ experience of knowledge about phosphate medication, both in terms of lack information from healthcare professionals, but also lack of motivation to find out their own knowledge. In addition, the study shows weaknesses regarding hemodialysis patient´s understanding of the importance of phosphate medication. Conclusion: The study shows that approximately half of the hemodialysis patients are capable to properly administer the phosphate-binding drugs. However, there are still shortcomings in some patients regarding the knowledge of phosphate-binding drugs.

Les « phosphate binding protein » (PBP) constituent une famille de protéines présentes de manière ubiquitaire chez les bactéries et plus marginalement chez les Eucaryotes. Impliquées dans l'import du phosphate extracellulaire chez les bactéries, les PBPs présentent un site de fixation du phosphate très bien caractérisé avec, notamment, une liaison hydrogène particulière nommée «low barrier hydrogen bond» (LBHB). Cette LBHB est impliquée dans la discrimination entre le phosphate et des anions proches chez les PBPs. Bien que cette discrimination semble nécessiter une haute conservation du site de fixation du phosphate, dans la nature différentes configurations sont observées. Au cours de ce travail, nous nous sommes intéressés à la PBP d'un organisme pathogène, C.perfringens qui présente un site de fixation alternatif. Avec, entre autre, une perte de la LBHB, cette PBP présente la plus faible capacité de discrimination testée à ce jour. Cette faible capacité de discrimination pourrait être liée au biotope de la bactérie ou bien à un phénomène d'adaptation fonctionnelle. D'autre part, certaines PBPs présentent des propriétés d'inhibition du VIH via l'étape de la transcription virale. Cependant, ces protéines sont particulièrement difficiles à produire en système hétérologue limitant l'étude fonctionnelle. Afin de lever ce verrou technique, nous avons développé une nouvelle méthodologie basée sur la phylogénie en vue de solubiliser notre modèle d'étude (HPBP). Nous avons obtenu un variant soluble de HPBP qui conserve ses activités antivirales permettant de débloquer les études fonctionnelles. / The "phosphate binding protein" constitutes a family of proteins ubiquitously found in Prokaryotes but also more sparsely distributed in Eukaryotes. Involved in phosphate import, PBPs exhibits a well-characterized phosphate binding site with a peculiar hydrogen bond called "low barrier hydrogen bond" (LBHB). This LBHB is involved in the unique discrimination properties of PBPs, capable of discriminating phosphate from other similar anions such as arsenate of sulfate. Albeit this high discriminating property needs a high conservation of the phosphate binding pocket, different configurations are observed in nature. Herein, we have been interested in a PBP from a human pathogen, Clostridium perfringens, which presents an alternative phosphate binding site. Exhibiting a loss of the LBHB, C.perfringens PBP is the least discriminating PBP isolated so far. This weak discrimination property might be related to the environment of C.perfringens or to a functional adaptation of the PBP. On the other hand, PBPs issued from eukaryotic tissues exhibit HIV inhibition properties via a step not yet targeted in current therapies, i.e. the transcription. However, these proteins are difficult to obtain from human tissues and their expression in heterologous system remains impossible. We have developed a new methodology based on phylogeny in order to solubilise our study model, HPBP. Thus, we have obtained a soluble variant of HPBP which conserves the HIV-inhibiting properties. This unique tool both allow to unlock functional studies and lead to a better understanding on how PBPs are capable of inhibiting HIV.

Occasionally, crystallisation of proteins works in mysterious ways! One might obtain crystals of a protein of unknown identity in place of the protein for which crystallisation experiments were performed. If the investigator is not aware of such possibilities, valuable time and resources might be lost in attempting to determine the structure of such proteins. Instances of non-target protein getting crystallised may not come to light at all or may be realised only when attempts to determine the structure completely fail by conventional procedures after collecting and processing the diffraction data. Usually, it is not possible to reproduce the crystals of the same protein as their occurrence is serendipitous. Such rare instances of crystallisation are probably caused by fluctuating environmental or crystallisation conditions and are not reproducible. It could also be due to contaminating microbes, which is more likely when the experimentalist is not well experienced. Therefore, experimental phasing of the data collected on serendipitously obtained crystals could be a challenging task. With the rapid increase in the number of structures deposited in the protein data bank (PDB), molecular replacement has become the method of choice for structure determination in macromolecular X-ray crystallography. This is due to the fact that it is possible to select a suitable phasing model for most target proteins based on their sequence information. However, if the identity of the target protein itself is uncertain, all attempts of structure determination using phasing models selected on the basis of target protein sequence-dependent search would fail. Sequence-independent ab initio phasing techniques such as ARCIMBOLDO (Meindl et al., 2012), which has recently become available, could provide leads only if the non-target protein is an all-α-protein and the associated diffraction data extends to a resolution better than 2 Å. Even then, the success rate with this technique is low. Hence, it becomes important to employ a sequence-independent method of structure determination for such mysteriously obtained crystals. This thesis reports crystal structures of proteins which are serendipitously crystallised using a large-scale application of Molecular Replacement (MR) technique (referred in this thesis as MarathonMR). This thesis also presents an evaluation of molecular replacement strategies for structure determination. The thesis begins with an overview of crystallographic methods of structure determination with an emphasis on the method of molecular replacement (Chapter 1). The most prominent of the results obtained in the course of these investigations pertains to a crystal obtained during routine crystallisation of a viral protein mutant in the year 2011. The cell parameters were different from cell constants of crystals obtained with other known viral protein mutants crystallised earlier in the same laboratory. Unfortunately, this crystal could not be reproduced in the same form in subsequent crystallisation trials. All attempts to determine the structure through conventional molecular replacement techniques using a combination of domains from a nearly identical virus coat protein protomer as the phasing model had failed. The data was shelved as “not-solvable” in late 2011. However, the crystal had diffracted to 1.9 Å and had excellent merging statistics. Therefore, the data was retrieved recently and additional attempts were made to determine the structure through phasing techniques that have become available recently. Techniques such as AMPLE (Bibby et al., 2013) and Rosetta (DiMaio, 2013), which use large-scale homology models coupled with molecular replacement, did not lead to meaningful solutions. A couple of helices identified by ARCIMBOLDO (Meindl et al., 2012) were neither correct (retrospectively) nor sufficient to determine the entire structure. Given the excellent merging statistics of the crystal data, there was significant motivation to determine the structure, though it meant developing a fresh protocol. It was at this time that we came across the work of Stokes-Rees and Sliz (2010) in which they had demonstrated that it is possible to determine structure of proteins of unknown identity by employing almost every known protein structure as a potential phasing model. The work reported in the thesis is a result of an earlier project to examine the relationship between properties of phasing models and the quality of target protein model generated through MR by employing large scale molecular replacement runs. This project was initiated because of the realisation that the recent explosion in crystallographic structural studies has resulted in near complete exploration of the “fold-space” of proteins and PDB now has a representative structure for most plausible folds of proteins. Some folds are highly represented in the PDB. Hence, it is likely that there would be at least one homologue in the PDB which could be used as a phasing model to successfully determine the structure of a protein of unknown identity if the diffraction dataset is of excellent quality. Hence, the single dataset which had diffracted to 1.9 Å resolution was used to develop a MarathonMR procedure for structure determination. MarathonMR procedure takes sequence-independent approach to structure determination and employs large-scale molecular replacement calculations to identify the closest homologue (in structural terms initially). This protocol is described in Chapter 2 (Materials and methods) of the thesis. Through MarathonMR, structure of the dataset which had remained unsolved for 5 years was finally determined. Nearly complete sequence of the polypeptide could be deduced by inspecting the electron density map due to the high resolution and quality of the map. The protein was found to be a phosphate binding protein from a soil bacterium Stenotrophomonas maltophilia (SmPBP). The way in which the structure was determined and possible explanations for the mysterious source of this protein which had crystallised instead of the target protein is discussed in Chapter 3. Though MarathonMR procedure was developed to solve a single dataset, it was soon realised that the same procedure could be applied to other similar datasets, all of which had diffracted to reasonable resolutions with good merging statistics but had remained unsolved for unknown reasons. Among such datasets, one of the datasets which was collected in 2007 and had diffracted to 2.3 Å resolution had cell parameters very close to that of SmPBP. Hence, a poly-alanine model of the structure of SmPBP, which was determined by then, was used as the phasing model to run molecular replacement and the structure was readily solved. It was surprising to note that SmPBP had crystallised serendipitously not once but twice, once in 2011 resulting in crystals that diffracted to 1.9 Å resolution and earlier in 2007 in crystals that diffracted to 2.3 Å resolution independently by two different investigators in the same laboratory. Both the structures are nearly identical and a comparison of these structures is presented in Chapter 4. Structure of SmPBP determined at 2.3 Å resolution by MarathonMR also corresponds to the dataset that had remained unsolved for the longest period of time (9 years). This success of structure determination after the lapse of such a long period emphasises the importance of carefully preserving X-ray diffraction data irrespective of its immediate outcome. In Chapter 5 of the thesis, another instance of non-target protein crystallisation, the structure of which was determined using the MarathonMR procedure is described. The crystal was obtained while carrying out crystallisation of mutants of a survival protein (SurE) expressed in Salmonella typhimurium when the bacterium is subjected to environmental or internal stresses. The original investigator had used the structure of SurE as the phasing model to determine structure of the mutant crystals and obtained a model with R and Rfree of 35% and 40%, respectively. However, the model did not refine further to lower R-factors suggesting that the solution obtained may not be correct. MarathonMR indicated that the fold of the crystallised protein could be similar to that of glycerol dehydrogenase. As SurE shares some fold similarity with one of the domains of GlyDH, the original investigator might have been able to achieve a limited success with R/Rfree factors of 35% and 40%, respectively. As the merging statistics for this diffraction data set was poor, the diffraction images were reprocessed in XDS program on Xia2 automated spot processing pipeline. The data statistics indicated merohedral twinning (14%). However, using appropriate parameters, it was possible to refine the structure obtained by MarathonMR to acceptable R/Rfree using the Refmac program. Four protomers were present in the crystal asymmetric unit (ASU). Non-crytsallographic symmetry averaging of electron density over these four molecules further improved the electron density. As the data was limited to 2.7 Å resolution, it was not possible to deduce the identity of every residue of the protein unambiguously based solely on the resulting electron density map. With the identity of the amino acids that could be deduced with certainty, it was clear that the protein belongs to glycerol dehydrogenase from a species of Enterobacteriacea family. Though a similar structure of glycerol dehydrogenase has been reported from Serratia, there are clear differences in many unambiguously determined residues which suggest that the protein is not from Serriatia. The protein has been named EnteroGlyDH as the source of the protein is likely to be from a species of Enterobacteriacea family. The structure of the protein, its biochemical implications and possible reasons for the serendipitous crystallisation of a non-target are discussed. Chapter 6 discusses the structure determination of an inorganic pyrophosphatase and catalytic domain of Succinyl transferase, the crystals of which had diffracted to 2.3 Å and 3.1 Å, respectively, but had remained unsolved. Neither of the datasets corresponds to the intended target proteins. The dataset corresponding to the protein whose structure was determined as that of an inorganic pyrophosphatase was provided by a colleague from a different laboratory in the Indian Institute of Science. It is interesting to note that the investigator had carried this dataset to one of the CCP4 workshops and had tried to determine the structure with the help of experts in the workshop. The attempts to determine its structure had however failed for reasons that are obvious now. The original investigator was unfortunately making efforts with an erroneous assumption on the identity of the target protein. As these enzymes are well studied, their structures and functions are briefly discussed. It is already well established that molecular replacement is being used with increasing frequency as the phasing technique when compared to other experimental phasing techniques. With the ever growing number of structures in the PDB, high population of certain folds and a near-plateau attained in the identification and growth of new folds, it is reasonable to expect that molecular replacement will be used even more frequently in the years to come. Therefore, for carrying out molecular replacement for a given diffraction dataset of a target protein, it is very likely that several homologous structures would be available in the PDB that could be used as potential phasing models. Hence, it becomes important to understand the influence of phasing model on the quality and accuracy of model generated through MR to achieve the best structure solution. To understand this relationship between phasing model and model obtained by MR protocol, re-determination of already known structures deposited in the PDB starting with their respective structure factors and various phasing models was initiated. Structures belonging to TIM beta/alpha-barrel (SCOPe ID: c.1) and Lysozyme-like (SCOPe ID: d.2) folds were chosen as targets. The structure of each target was re-determined serially starting with poly-alanine models of all available unique homologues as phasing models. Due to the multi-dimensional nature of this study, the results obtained were represented in a graphical form with nodes and edges. Detailed methodology of the work carried out and the data representation model are discussed in the Chapter 2 (Materials and methods). It was found that after a certain sequence identity cut-off, sequence identity between phasing model and target seems to have little influence on the quality and accuracy of the model generated through MR. Instead, other qualities of the phasing model such as Rfree and RSCC influence the quality of MR models. These results are discussed in Chapter 7. Learning from the work reported in this thesis are discussed in concluding chapter. The possible logical and programmatic upgrades to MarathonMR protocol and future path in which the relationship between phasing models and models generated through MR can be studied are discussed in Chapter 8 (Conclusion and future prospects).

Orientador: Andrea Balan Fernandes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T08:19:27Z (GMT). No. of bitstreams: 1 Pegos_VanessaRodrigues_D.pdf: 41543267 bytes, checksum: 0c6ab77fc1de1527fad27fbf8c4b1e70 (MD5) Previous issue date: 2015 / Resumo: Xanthomonas axonopodis pv. citri (X. citri) é o causador do cancro cítrico em diversas espécies de citrus, sobretudo, laranjas. As epidemias de cancro cítrico tem causado severas perdas econômicas à citricultura mundial uma vez que não há estratégias de combate efetiva contra essa bactéria no campo. Diversos estudos demonstraram a importância de genes para a patogênese de X. citri, mas ainda não foram investigados genes envolvidos na aquisição e no metabolismo de micronutrientes tais como o fosfato. X. citri conserva o sistema de transporte do tipo ABC de fosfato inorgânico codificado pelo óperon pstSCAB. Adicionalmente a bactéria possui dois outros operons oprO/phoX e phoBR, os quais codificam, respectivamente, uma porina de membrana externa e uma proteína periplasmática ligadora e o sistema dois componentes de sinalização celular, ambos integrantes do regulon de fosfato (regulon pho). Neste trabalho, estudamos a resposta destes operons à carência de fosfato, bem como o papel da proteína ligadora periplasmática PstS, por meio de análises proteômica, metabolômica, estruturais baseadas em cristalografia de raio-X e funcionais utilizando um mutante de X. citri portador de deleção no gene pstS (Xac::pstS). Os dados obtidos foram comparados entre as linhagens selvagem e mutante. Primeiramente evidenciamos que o sistema ABC de fosfato é ativado em carência do íon, incluindo um aumento de expressão de PhoX e PstS de 49 e 33 vezes, respectivamente, e que X. citri apresenta a maioria dos genes do regulon pho. PhoX e PstS são proteínas ligadoras de fosfato que partilham 70% de identidade de aminoácidos e são originadas de uma duplicação gênica. Na ausência de PstS, PhoX parece exercer a função de captação, mas não é capaz de recuperar todos os fenótipos da bactéria selvagem. Adicionalmente, ensaios de transporte de fosfato com as bactérias selvagem e mutante mostraram diferenças no transporte e que o sistema ABC permance constitutivo na linhagem mutante. A deleção de pstS também culminou no retardamento do crescimendo da bactéria em folhas de C. sinensis, mas não interferiu na adesão bacteriana e na produção da goma, estas sim, influenciadas diretamente pela concentração de fosfato no meio. Análises de metabolômica evidenciaram que a carência de fosfato induz mudanças nas rotas bioquímicas, sobretudo na linhagem mutante que utiliza da via das pentoses e do metabolismo do piruvato para a produção de ATP. Este é o primeiro trabalho que evidencia o papel do sistema ABC de transporte de fosfato nesta bactéria e que relaciona de uma forma multidisciplinar, o papel do íon e dos componentes do regulon pho na bactéria X. citri. Adicionalmente, uma vez que o sistema é bem conservado em outras espécies, os resultados obtidos servem como modelo para o gênero Xanthomonas / Abstract: ! Xanthomonas axonopodis pv. citri (X. citri) is the cause of citrus canker in several species of citrus, especially oranges. The citrus canker epidemics have caused severe economic losses to the citrus industry worldwide since no effective combat strategies against this bacterium. Several studies have demonstrated the importance of genes related to the pathogenesis of X. citri, but there is no studies about mechanisms of micronutrients acquisition such as phosphate. X. citri has an ATP-Binding Cassete transport system for inorganic phosphate encoded by pstSCAB operon. In addition, the bacterium has two other operons oprOphoX and phoBR, which encode respectively, an outer membrane porin and a periplasmic binding protein and two-components system. The three operons and other related genes are members of the phosphate regulon (pho regulon). In this work we studied the response of these operons in phosphate deprivation, and the role of periplasmic-binding protein PstS through proteomics analysis, metabolomics, crystallography and functionally based on a X. citri mutant deleted for pstS gene (Xac::pstS). Data were compared between wild type and mutant strains. We showed that the phosphate ABC system is activated during the ion depletion, including PstS and PhoX that showed increased levels of 49 and 30 times, respectively. In addition, we showed that X. citri displays most of the genes of the pho regulon. PhoX and PstS are phosphate binding proteins that share 70% amino acid identity and have origin from a gene duplication. In the absence of PstS, PhoX seems to complement the uptake function, but it is not able to recover all phenotypes of the wild type bacteria. Additionally, phosphate transport assays with wild type and mutant bacteria showed differences in transport and constitutivity of the ABCsystem in the mutant strain. The deletion of pstS also resulted in slowing of bacteria growth in Citrus sinensis leaves, but did not interfere with bacterial adhesion and gum production, two phenomena directly influenced by the phosphate concentration in the medium. Metabolomic analyzes showed that phosphate deprivation induces changes in biochemical pathways, especially the mutant strain that uses the pentose and pyruvate metabolism for ATP production. This is the first work that highlights the role of the ABC system for phosphate in this bacterium and that reveals in a multidisciplinary way, the role of the ion and the pho regulon components in the phytopathogenic bacterium. Additionally, once the system is well preserved in other species, the results serve as a model for the genus Xanthomonas / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular