Towards reverse engineering of Photosystem II: Synergistic Computational and Experimental Approaches

ABSTRACT Photosystem II (PSII) of oxygenic photosynthesis has the unique ability to photochemically oxidize water, extracting electrons from water to result in the evolution of oxygen gas while depositing these electrons to the rest of the photosynthetic machinery which in turn reduces CO2 to carbohydrate molecules acting as fuel for the cell. Unfortunately, native PSII is unstable and not suitable to be used in industrial applications. Consequently, there is a need to reverse-engineer the water oxidation photochemical reactions of PSII using solution-stable proteins. But what does it take to reverse-engineer PSII’s reactions? PSII has the pigment with the highest oxidation potential in nature known as P680. The high oxidation of P680 is in fact the driving force for water oxidation. P680 is made up of a chlorophyll a dimer embedded inside the relatively hydrophobic transmembrane environment of PSII. In this thesis, the electrostatic factors contributing to the high oxidation potential of P680 are described. PSII oxidizes water in a specialized metal cluster known as the Oxygen Evolving Complex (OEC). The pathways that water can take to enter the relatively hydrophobic region of PSII are described as well. A previous attempt to reverse engineer PSII’s reactions using the protein scaffold of E. coli’s Bacterioferritin (BFR) existed. The oxidation potential of the pigment used for the BFR ‘reaction centre’ was measured and the protein effects calculated in a similar fashion to how P680 potentials were calculated in PSII. The BFR-RC’s pigment oxidation potential was found to be 0.57 V, too low to oxidize water or tyrosine like PSII. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of iii tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. The results were used to develop a second generation of BFR-RC using a high oxidation pigment. The hypervalent phosphorous porphyrin forms a radical pair that can be observed using Transient Electron Paramagnetic Resonance (TR-EPR). Finally, the results from this thesis are discussed in light of the development of solar fuel producing systems

Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica EstruturalThe microbial plant cell wall degradation is one of the most important processes in the global turnover of atmospheric carbon dioxide. The work presented in this thesis addressed the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens, essential to the process of cellulose degradation, and aimed to study some of the components involved in their architecture (cohesins and dockerins) and efficiency (Carbohydrate-Binding Modules - CBMs). For this I used a combination of Nuclear Magnetic Resonance (NMR), X-ray crystallography and computer modeling techniques. My objective was to help rationalize the molecular determinants of specificity of CBMs, including the CtCBMs of families 11, 30 and 44, and the mechanisms of molecular recognition between cohesins and dockerins. In Chapter I, I present a general introduction to the theme of degradation of plant cell walls, with special attention to the cellulosome and its components. In Chapter II, I discuss the structural characteristics of the CtCBM11 based on the structures obtained by NMR at 25 and 50 °C and the structure obtained by crystallography. I found that although similar, the structures show some differences, particularly regarding the binding cleft area, which explains the negative results obtained by co-crystallization. In Chapter III and IV I study the molecular determinants of specificity in modules CtCBM11, 30 and 44, based on NMR and computer modeling data. I found that the atoms of the cellooligosaccharides most important for binding are the ones at positions 2 and 6 of the central units of the ligands. Moreover, I characterized the mechanisms responsible for selection and binding of these modules to various substrates. I established that binding occurs by a mechanism for conformational selection, where the topology of the residues of the protein, the conformation of the ligand and the number of glucose units, play a fundamental role. Chapters V and VI reveal the determination of the 3D structure of the cohesin-module X-dockerin complex of C. thermocellum and the cohesin-dockerin complex of B. cellulosolvens, respectively. Both complexes belong to the type II and their analysis allowed obtaining important information about the structural features that define the cohesin-dockerin interaction. The structure belonging to C. thermocellum revealed that the module X is essential for the stability of the complex. Moreover, for the first time the 3D structure of a cohesin-dockerin complex from B. cellulosolvens was determined. In this complex the dockerin is rotated 180º when compared to other complexes. This gives the cellulosome plasticity. In the final chapters, I present the NMR and X-ray crystallography techniques I used throughout the study. Finally, I draw some general conclusions about all the work done.Fundação para a Ciência e Tecnologia - SFRH/BD/35992/2007, and projects PTDC/QUI/68286/2006, PTDC/QUI-BIQ/100359/2008 and PTDC/BIA-PRO/103980/200

Molecular modelling of platelet endothelial cell adhesion molecule 1 and its interaction with glycosaminoglycans

The Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) has many functions including its roles in leukocyte extravasation as part of the inflammatory response, and in the maintenance of vascular integrity through its contribution to endothelial cell-cell adhesion. Various heterophilic ligands of PECAM-1 have been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this thesis. The three dimensional structure of the extracellular immunoglobulin (Ig)-domains of PECAM-1 was constructed using homology modelling and threading methods. Potential heparin/heparan sulfate binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein binding specificity and selectivity. It is predicted that two regions in PECAM-1 appear to bind heparin oligosaccharides. A high affinity binding region was located in Ig-domains 2 and 3 and a low affinity region was located in Ig-domains 5 and 6.These GAG binding regions are distinct from regions involved in PECAM-1 homophilic interactions. Docking of heparin fragments of different size revealed that fragments as small as a pentasaccharide appear to be able to bind to domains 2 and 3 with high affinity. Binding of longer heparin fragments suggests that key interactions can occur between six sulfates in a hexasaccharide with no further increase in binding affinity for longer fragments. Molecular dynamics simulations were also used to characterise and quantify the interactions of heparin fragments with PECAM-1. These simulations confirmed the existence of regions of high and low affinity for GAG binding and revealed that both electrostatic and van der Waals interactions determine the specificity and binding affinity of GAG fragments to PECAM-1. The simulations also suggested the existence of ‘open’ and ‘closed’ conformations of PECAM-1 around domains 2 and 3

Facettes de glycobioinformatique (applications à l'étude des interactions protéines-sucres)

Le travail décrit dans ce manuscrit rassemble les résultats obtenus au cours de ma thèse de doctorat. Ils s'inscrivent dans le domaine de la glycobioinformatique. Ils ont impliqué des développements de bases de données structurales et des applications en modélisation moléculaire des interactions protéines-sucres. Les méthodes de modélisation moléculaire ont été utilisées dans la reconstruction et dans la prédiction des structures tridimensionnelles de polysaccharides et d'oligosaccharides, ces dernières étant également établies par une approche de type haut-débit par application d'un algorithme génétique à des fins de minimisation énergétique. Les données ainsi générées ont été organisées sous la forme de bases de données relationnelles, proprement annotées (PolySca3DB et BiOligo) qui sont en libre accès pour consultation sur internet. Ces méthodes de modélisation moléculaire ont été appliquées à la caractérisation, par RMN en solution, des conformations de basse énergie d'une souche pathogène d'un polysaccharide de la bactérie E. coli. D'autres bactéries pathogènes de type gram négatif, interagissent avec des oligosaccharides par l'intermédiaire de protéines secrétées, telles que des lectines. Nous avons testé, au travers de l'utilisation de méthodes d'amarrage moléculaire, la possibilité d'identifier de manière automatique, la nature de ces interactions, en prenant comme cibles des épitopes oligosaccharidiques fucosylés. Les résultats de ces recherches ont été comparés, de manière critique, à ceux issus de l'application de bio-puces à sucres et de calorimétrie isotherme de titration. Les conclusions et perspectives de ces travaux sont présentées dans un article de revue consacré à l'application des méthodes de chimie computationnelle dans l'étude des interactions protéines-glucides qui viennent compléter l'arsenal des outils dédiés au champs de recherche couvert par la glycobiologie structurale et moléculaire.This thesis presents an account of two important facets of glycobioinformatics, comprising database development and molecular modeling of 3D structures of carbohydrates alongside the simulation of protein-carbohydrate interactions. Classical molecular modeling techniques were used to reconstruct 3D polysaccharide structures from experimentally determined atomic coordinates, or known starting points about their structures were used as guidelines to model them. A genetic algorithm search was employed as a high-throughput technique to characterize low energy conformers of bioactive oligosaccharides. The data generated were organized into two open-access relational databases, namely, PolySac3DB and BiOligo, for use by the scientific community. The validation of the molecular techniques used were performed using solution phase NMR experiments on four entero aggregative pathogenic E. coli strains, and were found to be robust and realistic. Further, the impact of the presentation of human fucosylated oligosaccharide epitopes to lectins from opportunistic gram negative bacteria, was investigated in a screening study using molecular docking studies, which could help in evaluating the feasibility of using automated docking procedures in such instances as well as deciphering binding data from glycan array experiments and also correlated to isothermal calorimetry data. On comparison with high-resolution experimental crystal complexes, automated docking was found to delineate the present level of applicability, while emphasizing the need of constant monitoring and possible filtering of the results obtained. Finally, a review of the present status of the computational aspects of protein-carbohydrate interaction studies is discussed in the perspectives of using molecular modeling and simulation studies to probe this aspect of molecular and structural glycobiology.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Understanding The Function Of Conserved Variations In The Catalytic Loops Of Fungal Glycoside Hydrolase Family 12

Enzymes that cleave the xyloglucan backbone at unbranched glucose residues have been identified in GH families 5, 7, 12, 16, 44, and 74. Fungi produce enzymes that populate 20 of 22 families that are considered critical for plant biomass deconstruction. We searched for GH12-encoding genes in 27 Eurotiomycetes genomes. After analyzing 50 GH12-related sequences, the conserved variations of the amino acid sequences were examined. Compared to the endoglucanases, the endo-xyloglucanase-associated YSG deletion at the negative subsites of the catalytic cleft with a SST insertion at the reducing end of the substrate-binding crevice is highly conserved. In addition, a highly conserved alanine residue was identified in all xyloglucan-specific enzymes, and this residue is substituted by arginine in more promiscuous glucanases. To understand the basis for the xyloglucan specificity displayed by certain GH12 enzymes, two fungal GH12 endoglucanases were chosen for mutagenesis and functional studies: an endo-xyloglucanase from Aspergillus clavatus (AclaXegA) and an endoglucanase from A. terreus (AtEglD). Comprehensive molecular docking studies and biochemical analyses were performed, revealing that mutations at the entrance of the catalytic cleft in AtEglD result in a wider binding cleft and the alteration of the substrate-cleavage pattern, implying that a trio of residues coordinates the interactions and binding to linear glycans. The loop insertion at the crevice-reducing end of AclaXegA is critical for catalytic efficiency to hydrolyze xyloglucan. 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