As aminoacil tRNA sintetases (AaRSs) são enzimas essenciais na síntese de proteínas assegurando a correta relação entre os aminoácidos e seus tRNA cognatos. O genoma mitocondrial dos tripanossomatídeos perdeu os genes codificantes dos tRNAs, assim os tRNA mitocondriais são codificados no núcleo e importados do citoplasma. O código genético do kinetoplasto desvia do código genético pela utilização do códon de terminação UGA para a decodificação do códon do triptofano. Um único gene codificando o tRNATrp(CCA) observado no genoma de Leismania é responsável pela incorporação do aminoácido triptofano durante a síntese proteíca na mitocôndria. Para decodificar os dois códons do Trp (UGA e UGG) a base na posição 34 do tRNATrp(CCA) passa por um evento de editoração, convertendo o ribunuclotídeo C34 em U34, produzindo o tRNATrp(UCA) capaz de decodificar o códon UGA. Nesse trabalho foram caracterizadas duas triptofanil tRNA sintetases de Leishmania major. De acordo com experimentos de ?western blotting? e análises ?in silico? das seqüências de aminoácidos, uma enzima tem localização citoplasmática (LmTrpRS1) enquanto a outra mitocondrial (LmTrpRS2). Os mRNAs dos dois genes foram definidos por experimentos de 5? e 3? RT-PCR. As duas enzimas foram clonadas em diversos vetores de expressão procariotos e eucariotos. A LmTrpRS1 foi obtida somente na fração insolúvel, já a LmTrpRS2 foi obtida na fração solúvel quando clonada no vetor de expressão pET28a. Esta porém mostrou-se instável precipitando rapidamente após sua purificação. Os ensaios enzimáticos realizados com a mesma mostraram que ela é capaz de reconhecer os tRNAsTrp editado e não editado. Modelagem molecular por homologia com as duas proteínas foi realizada usando a proteína citoplasmática humana como molde, para estudar a interação entre a proteína e o tRNATrp. Xylella fastidiosa é um bactéria gram negativa limitada ao xilema, responsável por um grande número de doenças economicamente importantes, como a doença de Pierces em videiras, Clorose variegata do Citrus (CVC) e a doença da requeima das folhas em outras plantas incluindo, amendoeira, ameixeira, louro, amoreira e café. Em todos os casos a X. fastidiosa afeta o xylema da planta causando redução na produção de frutos. Nesse trabalho nós mostramos a estrutura da Xylellaína, uma cisteíno protease desse patógeno. A estrutura foi resolvida por dispersão anômala a um único comprimento de onda, utilizando cristais de xylellaína selenometionina substituídos. A estrutura da Xylellaína foi refinada até 1,65 Å de resolução, mostrando enovelamento similar às proteínas da família da papaína, porém algumas características interessantes como uma região N-terminal composta por 38 aminoácidos cobrindo o sulco ativo da enzima, um intrigante ribonucleotídeo encontrado fora do sítio ativo da enzima e um ?loop? semelhante ao ?loop? de oclusão presente na catepsina B. / The aminoacyl tRNA synthetases (aaRSs) are essential enzymes in protein synthesis that ensure the correct match between amino acids and their cognate tRNAs. The mitochondrial (kinetoplast) genome of trypanossomatids lacks tRNA genes, and therefore nucleus-encoded tRNAs are imported from the cytoplasm, the kinetoplast genetic code deviates from the universal code in that UGA instead of UGG encodes for tryptophan. A single nucleus-encoded tRNATrp(CCA) is responsible for Trp insertion during organellar protein synthesis. To decode both Trp codons (UGA and UGG), tRNATrp(CCA) undergoes a single C to U editing event at position 34 of the anticodon yielding to versions of the tRNA in the mitochondria with anticodon CCA and UCA, permitting UGA decoding. This work have characterized two Leishmania major tryptophanyl-tRNA synthetase, acording western blotting experiments and ?in silico? sequence analisis one of cytoplasmatic localization (LmTrpRS1) and another from mitochondria localization (LmTrpRS2). The mature mRNA transcripts for both genes were defined by 5? and 3? RT-PCR. Both enzymes were cloned into several expressions vectors. LmTrpRs1 was obtained as an insoluble protein and LmTrpRs2 expressed into the soluble fraction in pET28a expression system. LmTrpRS2 protein, however, is unstable precipitating shortly after purification. The enzymatic assay showed that this enzyme is able to recognize both tRNATrp. Molecular modeling for LmTrpRS1 and LmTrpRS2 were constructed using the cytoplasmatic human tryptophanyl tRNA synthetase as a model, to study the interaction between proteins and tRNATrp. Xylella fastidiosa is a xylem-limited, gram-negative bacteria responsible for a large number of economically important plant diseases, such as Pierces disease in grapevines, citrus variegated chlorosis (CVC) in sweet oranges and leaf scorch diseases in other plants, including almond, plum, oleander, mulberry and coffee. In all cases, X. fastidiosa infects the plant xylem and impairs fruit production. Here, we report the crystal structure of xylellain, a cystein protease from X. fastidiosa. The structure was solved by single-wavelength anomalous dispersion (SAD) using seleno-methionine containing xylellain crystals. The final structure of Xylellaína was refined against the best native data set (1.65 Å) showing R/Rfree= 17/21. Xylellain shares fold similar to Papain like Family, but contains some interesting features, like a 38 N-terminal tail covering the active site cleft; one intriguing ribonucleotide found outside the active site and one loop that resemble the ocluding loop from cathepsin B.

As aminoacil tRNA sintetases (AaRSs) são enzimas essenciais na síntese de proteínas assegurando a correta relação entre os aminoácidos e seus tRNA cognatos. O genoma mitocondrial dos tripanossomatídeos perdeu os genes codificantes dos tRNAs, assim os tRNA mitocondriais são codificados no núcleo e importados do citoplasma. O código genético do kinetoplasto desvia do código genético pela utilização do códon de terminação UGA para a decodificação do códon do triptofano. Um único gene codificando o tRNATrp(CCA) observado no genoma de Leismania é responsável pela incorporação do aminoácido triptofano durante a síntese proteíca na mitocôndria. Para decodificar os dois códons do Trp (UGA e UGG) a base na posição 34 do tRNATrp(CCA) passa por um evento de editoração, convertendo o ribunuclotídeo C34 em U34, produzindo o tRNATrp(UCA) capaz de decodificar o códon UGA. Nesse trabalho foram caracterizadas duas triptofanil tRNA sintetases de Leishmania major. De acordo com experimentos de ?western blotting? e análises ?in silico? das seqüências de aminoácidos, uma enzima tem localização citoplasmática (LmTrpRS1) enquanto a outra mitocondrial (LmTrpRS2). Os mRNAs dos dois genes foram definidos por experimentos de 5? e 3? RT-PCR. As duas enzimas foram clonadas em diversos vetores de expressão procariotos e eucariotos. A LmTrpRS1 foi obtida somente na fração insolúvel, já a LmTrpRS2 foi obtida na fração solúvel quando clonada no vetor de expressão pET28a. Esta porém mostrou-se instável precipitando rapidamente após sua purificação. Os ensaios enzimáticos realizados com a mesma mostraram que ela é capaz de reconhecer os tRNAsTrp editado e não editado. Modelagem molecular por homologia com as duas proteínas foi realizada usando a proteína citoplasmática humana como molde, para estudar a interação entre a proteína e o tRNATrp. Xylella fastidiosa é um bactéria gram negativa limitada ao xilema, responsável por um grande número de doenças economicamente importantes, como a doença de Pierces em videiras, Clorose variegata do Citrus (CVC) e a doença da requeima das folhas em outras plantas incluindo, amendoeira, ameixeira, louro, amoreira e café. Em todos os casos a X. fastidiosa afeta o xylema da planta causando redução na produção de frutos. Nesse trabalho nós mostramos a estrutura da Xylellaína, uma cisteíno protease desse patógeno. A estrutura foi resolvida por dispersão anômala a um único comprimento de onda, utilizando cristais de xylellaína selenometionina substituídos. A estrutura da Xylellaína foi refinada até 1,65 Å de resolução, mostrando enovelamento similar às proteínas da família da papaína, porém algumas características interessantes como uma região N-terminal composta por 38 aminoácidos cobrindo o sulco ativo da enzima, um intrigante ribonucleotídeo encontrado fora do sítio ativo da enzima e um ?loop? semelhante ao ?loop? de oclusão presente na catepsina B. / The aminoacyl tRNA synthetases (aaRSs) are essential enzymes in protein synthesis that ensure the correct match between amino acids and their cognate tRNAs. The mitochondrial (kinetoplast) genome of trypanossomatids lacks tRNA genes, and therefore nucleus-encoded tRNAs are imported from the cytoplasm, the kinetoplast genetic code deviates from the universal code in that UGA instead of UGG encodes for tryptophan. A single nucleus-encoded tRNATrp(CCA) is responsible for Trp insertion during organellar protein synthesis. To decode both Trp codons (UGA and UGG), tRNATrp(CCA) undergoes a single C to U editing event at position 34 of the anticodon yielding to versions of the tRNA in the mitochondria with anticodon CCA and UCA, permitting UGA decoding. This work have characterized two Leishmania major tryptophanyl-tRNA synthetase, acording western blotting experiments and ?in silico? sequence analisis one of cytoplasmatic localization (LmTrpRS1) and another from mitochondria localization (LmTrpRS2). The mature mRNA transcripts for both genes were defined by 5? and 3? RT-PCR. Both enzymes were cloned into several expressions vectors. LmTrpRs1 was obtained as an insoluble protein and LmTrpRs2 expressed into the soluble fraction in pET28a expression system. LmTrpRS2 protein, however, is unstable precipitating shortly after purification. The enzymatic assay showed that this enzyme is able to recognize both tRNATrp. Molecular modeling for LmTrpRS1 and LmTrpRS2 were constructed using the cytoplasmatic human tryptophanyl tRNA synthetase as a model, to study the interaction between proteins and tRNATrp. Xylella fastidiosa is a xylem-limited, gram-negative bacteria responsible for a large number of economically important plant diseases, such as Pierces disease in grapevines, citrus variegated chlorosis (CVC) in sweet oranges and leaf scorch diseases in other plants, including almond, plum, oleander, mulberry and coffee. In all cases, X. fastidiosa infects the plant xylem and impairs fruit production. Here, we report the crystal structure of xylellain, a cystein protease from X. fastidiosa. The structure was solved by single-wavelength anomalous dispersion (SAD) using seleno-methionine containing xylellain crystals. The final structure of Xylellaína was refined against the best native data set (1.65 Å) showing R/Rfree= 17/21. Xylellain shares fold similar to Papain like Family, but contains some interesting features, like a 38 N-terminal tail covering the active site cleft; one intriguing ribonucleotide found outside the active site and one loop that resemble the ocluding loop from cathepsin B.

La methionyl-ARNt synthétase (MetRS) de Saccharomyces cerevisiae aminoacyle les ARNt méthionine initiateur et élongateur (ARNtiMet et ARNteMet), mais possède également des fonctions atypiques. Nous avons montré que la MetRS rejoint le noyau durant la transition diauxique afin de réguler la transcription des gènes nucléaires des complexes III et V de la chaîne respiratoire mitochondriale. Pour ce faire, la MetRS possède au moins deux signaux de localisation nucléaire (NLS) dans sa séquence, l’un se situant dans les 55 premiers acides aminés (aa) et le second, au delà de la partie N-terminale lui permettant de recruter les sous-unités Rpb4 et Rpb7 de l’ARN pol II. Nous avons montré qu’en fermentation, la MetRS est clivée entre le 114ème et le 132ème aa et que cette forme clivée est essentielle à la viabilité des cellules, puisqu’un variant non clivé (MetRSK11A) ne permet pas la croissance. Nous avons surproduit et purifié un mutant de la MetRS clivée (MetRSΔ142) et montré que ce variant est plus efficace pour l’aminoacylation de l’ARNtiMet que la forme entière de MetRS. Ainsi, notre étude suggère que chez S. cerevisiae, la forme longue de MetRS cytoplasmique permet l’aminoacylation de l’ARNteMet, la forme longue de MetRS nucléaire régule la transcription, et la forme clivée de MetRS nucléaire et cytoplasmique permet l’aminoacylation de l’ARNtiMet / Methionyl-tRNA synthetase (MetRS) is the enzyme in charge of aminocylation of tRNA methionine initiator and elongator (tRNAiMet et tRNAeMet), but also displays atypical functions in Saccharomyces cerevisiae. In the present work, we showed that MetRS is imported to the nucleus during the diauxic shift in order to regulate transcription of genes coding for the complexes III and V subunits of the mitochondrial respiratory chain. To do so, MetRS harbors at least two nuclear localization signals (NLS), located within the 55 first aminoacids (NLS1) and beyond the N-terminal part (NLS2). The N- terminal part is responsible for the recruitment of RNA pol II subunits Rpb4 and Rpb7. We also showed that MetRS is cleaved through the 114th and the 132nd aminoacid during fermentation and that the proteolysed form is essential for the viability of the cell, since a mutant of MetRS which is not cleaved (MetRSK11A) did not allows the growth. We showed that an overproduced and purified a mutant representative of the cleaved form (MetRSΔ142) is more efficient for tRNAiMet aminoacylation than the full length MetRS. Thus, our study suggests that in S. cerevisiae, the cytoplasmic full length MetRS aminoacylates tRNAeMet, the nuclear full length MetRS regulates genes transcription, and the cytoplasmic and nuclear cleaved MetRS aminoacylates the tRNAiMet.

Les complexes multisynthétasiques (MSC) sont des complexes multi-protéiques identifiés dans un grand nombre d’organismes pro- et eucaryotes. Ils impliquent des protéines d’assemblages et des aminoacyl-ARNt synthétases (aaRSs), responsables de l’aminoacylation de leurs ARNts homologues au cours de la traduction. La taille et la composition des MSC varient selon les organismes, et le rôle de ces complexes n’est pas encore totalement compris. Il semblerait néanmoins que chez les eucaryotes, l’accrétion en complexe soit une stratégie mise en oeuvre par les cellules pour empêcher les aaRSs d’assurer des fonctions additionnelles. Chez S.cerevisiae,nous montrons que la dynamique du complexe AME, composé de la méthionyl- et de la glutamyl-ARNt synthétase (MRS et ERS) ainsi que de la protéine d’ancrage Arc1p, est dépendante du métabolisme de la levure. En respiration la MRS joue le rôle de facteur de transcription et régule l’expression des gènes nucléaires du complexe III et V de la chaîne respiratoire, tandis que l’ERS active la traduction mitochondriale. Cette étude montre que la relocalisation synchrone est primordiale pour l’adaptation des cellules au métabolisme respiratoire. / Multisynthetase complexes (MSC) are complexes made of several proteins and were identified in a wide variety of organisms from pro- to eukaryotes. They are usually made of assembly factors and aminoacyl-tRNA synthetases (aaRSs), which are responsible for the aminoacylation of their corresponding tRNAs during translation. Depending on the organisms, size and composition of these complexes differ greatly and their role is not fully understood yet. Although it seems that in eukaryotes, accretions of aaRSs into MSC prevent aaRSs to perform their additional functions. In the yeast Saccharomyces cerevisiae, we show that the dynamic of the AME complex, made of the méthionyl- and glutamyl-tRNA synthetases (MRS and ERS) and the assembly protein Arc1p is linkedto yeast metabolism. In respiration, MRS is imported in the nucleus to act as a transcription factor and regulates the expression of nuclear genes belonging to complex III and V of the respiratory chain, while ERS is imported in mitochondria to activate translation. This study shows that synchronous relocation of both aaRSs is crucial for yeast cells to adapt to respiratory metabolism.

La glycyl-ARNt synthétase humaine (GRS) est une enzyme clé dans la traduction des protéines dans le cytosol et la mitochondrie. Chez l’Homme, des mutations de la GRS conduisent à la neuropathie périphérique Charcot-Marie-Tooth (CMT). Bien que l’activité de la GRS soit ubiquitaire, les mutations associées à la CMT n’affectent que les nerfs périphériques, suggérant un rôle supplémentaire de la GRS dans les neurones. Pour comprendre ce rôle, nous avons d’abord élucidé le mécanisme particulièrement complexe qui contrôle l’expression de la GRS mitochondriale et cytosolique à partir du même gène. Nous avons identifié deux ARNm : un codant pour les deux enzymes ; et un autre plus long qui contient une IRES fonctionnelle et un uORF. Cet ARNm complexe, ne génère que la GRS cytosolique et montre que son expression et localisation sont étroitement contrôlées. De plus, nous avons montré une distribution particulière de la GRS dans des neurones, qui est un premier indice sur un rôle non canonique. / Human Glycyl-tRNA synthetase (GRS) is a housekeeping enzyme with a key role in protein synthesis, both in the cytosol and the mitochondria. In human, mutations in GRS cause the Charcot-Marie-Tooth (CMT) peripheral neuropathy. Though GRS activity is required in all cells, the CMT-associated mutations affect only the peripheral nervous system, suggesting an additional non canonical role.To understand how GRS is involved in CMT pathology, we first elucidated the original post-transcriptional regulatory mechanism that controls the expression of both the mitochondrial and the cytosolic GRS from a single gene. We identified two mRNA isoforms: one coding for both enzymes; and a longer one containing a functional IRES and an uORF encoding only the cytosolic GRS, evidence that expression and localization of human GRS are tightly controlled. Furthermore, we found a particular Ca2+ dependant distribution of GRS in neurons, giving us a first clue about a potential non-canonical role in neurons.

Les ARNt sont des molécules adaptatrices reliant l'information génétique de l’ARN messagers à la séquence d'acides aminés primaire des protéines. Les ARNt ont une structure typique, appelée "feuille de trèfle". Certains ARNt mitochondriaux montrent une forte dérivation de cette structure. Un cas extrême peut être observé dans les mitochondries du nématode R. culicivorax. Cette étude vise la caractérisation fonctionnelle de ces ARNt «bizarres» et de définir leurs propriétés structurales et leur fonctionnalité avec des protéines partenaires telles que les CCAses et les aminoacyl-ARNt synthetases. Ce travail révèle que les ARNt sans bras forment une structure secondaire en forme d'épingle à cheveux et que leurs structures 3D présentent une grande flexibilité intrinsèque. Les tests initiaux n’ont pas démontré l'activité d'aminoacylation. Cependant, les ARNt sans bras représentent des molécules fonctionnelles pour le CCAse, indiquant des adaptations de l’enzyme aux ARNt sans bras. / TRNAs are adapter molecules linking the genetic information of messenger RNAs with the primary amino acid sequence of proteins. tRNAs have a typical cloverleaf-like secondary structure. Some mitochondrial tRNAs show a high derivation from this canonical tRNA structure. An extreme case of structural truncations can be observed in mitochondria of the nematode R. culicivorax. This study aims the functional characterization of such “bizarre” tRNAs in defining their structural properties and their functionality with interacting partner proteins such as CCA-adding enzymes and aminoacyl-tRNA synthetases. This work reveals that armless tRNAs form a hairpin-shaped secondary structure. 3D structures exhibit a high intrinsic flexibility. Initial tests could not demonstrate aminoacylation activity. However, armless tRNAs represent functional molecules for CCA-incorporation, indicating adaptations of CCA-adding enzymes to armless tRNAs.

No description available.

Proteins—polymers of amino acids—are a major class of biomolecules whose myriad functions facilitate many crucial biological processes. Accordingly, human control over these biological processes depends upon the ability to study, produce, and modify proteins. One innovative tool for accomplishing these aims is cell-free protein synthesis (CFPS). This technique, rather than using living cells to make protein, simply extracts the cells' natural protein-making machinery and then uses it to produce protein in vitro. Because living cells are no longer involved, scientists can freely adapt the protein production environment in ways not otherwise possible. However, improved versatility and yield of CFPS protein production is still the subject of considerable research. This work focuses on two ideas for furthering that research.The first idea is the adaptation of CFPS to make proteins containing unnatural amino acids. Unnatural amino acids are not found in natural biological proteins; they are synthesized artificially to possess useful properties which are then conferred upon any protein made with them. However, current methods for incorporating unnatural amino acids do not allow incorporation of more than one type of unnatural amino acid into a single protein. This work helps lay the groundwork for the incorporation of different unnatural amino acid types into proteins. It does this by using modified aminoacyl-tRNA synthetases (aaRSs), which are key components in CFPS, to be compatible with unnatural amino acids. The second idea is the preservation of DNA templates from enzyme degradation in CFPS. Among the advantages of CFPS is the option of using linear expression templates (LETs) in place of plasmids as the DNA template for protein production. Because LETs can be produced more quickly than plasmids can, using LETs greatly reduces the time required to obtain a DNA template for protein production. This renders CFPS a better candidate for high-throughput testing of proteins. However, LETs are more susceptible to enzyme-mediated degradation than plasmids are, which means that LET-based CFPS protein yields are lower than plasmid-based CFPS yields. This work explores the possibility of increasing the protein yield of LET-based CFPS by addition of sacrificial DNA, DNA which is not used as a protein-making template but which is degraded by the enzymes in place of the LETs.

The flow of genetic information from genes to proteins is mediated through proteins which interact with the nucleic acids at several stages to successfully transmit the information from the nucleus to the cell cytoplasm. Unlike in the case of protein-protein interactions, the principles behind protein-nucleic acid interactions are still not very (Pabo and Nekludova, 2000) and efforts are still underway to arrive at the basic principles behind the specific recognition of nucleic acids by proteins (Prabakaran et al., 2006). This is mainly due to the innate complexity involved in recognition of nucleotides by proteins, where, even within a given family of DNA binding proteins, different modes of binding and recognition strategies are employed to suit their function (Luscomb et al., 2000). Such difficulties have also not made possible, a thorough classification of DNA/RNA binding proteins based on the mode of interaction as well as the specificity of recognition of the nucleotides. The availability of a large number of structures of protein-nucleic acids complexes (albeit lesser than the number of protein structures present in the PDB) in the past few decades has provided the knowledge-base for understanding the details behind their molecular mechanisms (Berman et al., 1992). Previously, studies have been carried out to characterize these interactions by analyzing specific non-covalent interactions such as hydrogen bonds, van der Walls, and hydrophobic interactions between a given amino acid and the nucleic acid (DNA, RNA) in a pair-wise manner, or through the analysis of interface areas of the protein-nucleic acid complexes (Nadassy et al., 1998; Jones et al., 1999). Though the studies have deciphered the common pairing preferences of a particular amino acid with a given nucleotide of DNA or RNA, there is little room for understanding these specificities in the context of spatial interactions at a global level from the protein-nucleic acid complexes. The representation of the amino acids and the nucleotides as components of graphs, and trying to explore the nature of the interactions at a level higher than exploring the individual pair-wise interactions, could provide greater details about the nature of these interactions and their specificity. This thesis reports the study of protein-nucleic interactions using graph and network based approaches. The evaluation of the parameters for characterizing protein-nucleic acid graphs have been carried out for the first time and these parameters have been successfully employed to capture biologically important non-covalent interactions as clusters of interacting amino acids and nucleotides from different protein-DNA and protein-RNA complexes. Graph and network based approaches are well established in the field of protein structure analysis for analyzing protein structure, stability and function (Kannan and Vishveshwara, 1999; Brinda and Vishveshwara, 2005). However, the use of graph and network principles for analyzing structures of protein-nucleic acid complexes is so far not accomplished and is being reported the first time in this thesis. The matter embodied in the thesis is presented as ten chapters. Chapter 1 lays the foundation for the study, surveying relevant literature from the field. Chapter 2 describes in detail the methods used in constructing graphs and networks from protein-nucleic acid complexes. Initially, only protein structure graphs and networks are constructed from proteins known to interact with specific DNA or RNA, and inferences with regard to nucleic acid binding and recognition were indirectly obtained . Subsequently, parameters were evaluated for representing both the interacting amino acids and the nucleotides as components of graphs and a direct evaluation of protein-DNA and Protein-RNA interactions as graphs has been carried out. Chapter 3 and 4 discuss the graph and network approaches applied to proteins from a dataset of DNA binding proteins complexed with DNA. In chapter 3, the protein structure graphs were constructed on the basis of the non-covalent interactions existing between the side chains of amino acids. Clusters of interacting side chains from the graphs were obtained using the graph spectral method. The clusters from the protein-DNA interface were analyzed in detail for the interaction geometry and biological importance (Sathyapriya and Vishveshwara, 2004). Chapter 4 also uses the same dataset of DNA binding proteins, but a network-based approach is presented. From the analysis of the protein structure networks from these DNA binding proteins, interesting observations relating the presence of highly connected nodes(or hubs) of the network to functionally important amino acids in the structure, emerged. Also, the comparison between the hubs identified from the protein-protein and the protein-DNA interfaces in terms of their amino acid composition and their connectivity are also presented (Sathyapriya and Vishveshwara, 2006) Chapter 5 and 6 deal with the graph and network applications to a specific system of protein-RNA complex (aminoacyl-tRNA synthetases) to gain insights into their interface biology based on amino acid connectivity. Chapter 5 deals with a dataset of aminoacyl-tRNA synthetase (aaRS) complexes obtained with various ligands like ATP, tRNA and L-amino acids. A graph based identification of side chain clusters from these ligand-bound aaRS structures has highlighted important features of ligand-binding at the catalytic sites of the two structurally different classes of aaRS (Class I and Class II). Side chain clusters from other regions of aaRS such as the anticodon binding region and the ligand-activation sites are discussed. A network approach is used in a specific system of aaRS(E.coli Glutaminyl-tRNA synthetase (GlnRS) complexed with its ligands, to specifically understand the effects of different ligand binding., in chapter 6. The structure networks of E.coli GlnRS in the ligand-free and different ligand-bound states are constructed. The ligand-free and the ligand-bound complexes are compared by analyzing their network properties and the presence of hubs to understand the effect of ligand-binding. These properties have elegantly captured the effects of ligand-binding to the GlnRS structure and have also provided an alternate method for comparing three dimensional structures of proteins in different ligand-bound states (Sathyapriya and Vishveshwara, 2007). In contrast to protein structure graphs (PSG), both the interacting amino acids and nucleotides (DNA/RNA) form the components of the protein-nucleic acid graphs (PNG) from protein-nucleic acid complexes. These graphs are constructed based on the non-covalent interactions existing between the side chains of the amino acids and nucleotides. After representing the interacting nucleotides and amino acids as graphs, clusters of the interacting components are identified. These clusters are the strongly interacting amino acids and nucleotides from the protein-nucleic acid complexes. These clusters can be generated at different strengths of interaction between the amino acid side chain and the nucleotide (measured in terms of its atomic connectivity) and can be used for detecting clusters of non-specific as well as specific interactions of amino acids and nucleotides. Though the methodology of graph construction and cluster identification are given in chapter 2, the details of the parameters evaluated for constructing PNG are given in chapter 7. Unlike in the previous chapters, the succeeding chapters deal exclusively with results that are obtained from the analyses of PNG. Two examples of obtaining clusters from a PNG are given, one each for a protein-DNA and a protein-RNA complex. In the first example, a nucleosome core particle is subjected to the graph based analysis and different clusters of amino acids with different regions of the DNA chain such as phosphate, deoxyribose sugar and the base are identified. Another example of aminoacyl-tRNA synthetase complexed with its cognate tRNA is used to illustrate the method with a protein-RNA complex. Further, the method of constructing and analyzing protein-nucleic acid graphs has been applied to the macromolecular machinery of the pre-translocation complex of the T. thermophilus 70S ribosome. Chapter 8 deals exclusively with the results identified from the analysis of this magnificent macromolecular ensemble. The availability of the method that can handle interactions between both amino acids and the nucleotides of the protein-nucleic acid complexes has given us the basis fro evaluating these interactions in a level higher than that of analyzing pair-wise interactions. A study on the evaluation of short hydrogen bonds(SHB) in proteins, which does not fall under the realm of the main objective of the thesis, is discussed in the Chapter 9. The short hydrogen bonds, defined by the geometrical distance and angle parameters, are identified from a non-redundant dataset of proteins. The insights into their occurrence, amino acid composition and secondary structural preferences are discussed. The SHB are present in distinct regions of protein three-dimensional structures, such that they mediate specific geometrical constraints that are necessary for stability of the structure (Sathyapriya and Vishveshwara, 2005). The significant conclusions of various studies carried out are summarized in the last chapter (Chapter 10). In conclusion, this thesis reports the analyses performed with protein-nucleic acid complexes using graph and network based methods. The parameters necessary for representing both amino acids and the nucleotides as components of a graph, are evaluated for the first time and can be used subsequently for other analyses. More importantly, the use of graph-based methods has resulted in considering the interaction between the amino acids and the nucleotides at a global level with respect to their topology of the protein-nucleic acid complexes. Such studies performed on a wide variety of protein-nucleic acid complexes could provide more insights into the details of protein-nucleic acid recognition mechanisms. The results of these studies can be used for rational design of experimental mutations that ascertain the structure-function relationships in proteins and protein-nucleic acid complexes.

Aminoacyl-tRNA synthetases (aaRSs) are at the center of the question of the origin of life and are essential proteins found in all living organisms. AARSs arose early in evolution to interpret genetic code and are believed to be a group of ancient proteins. They constitute a family of enzymes integrating the two levels of cellular organization: nucleic acids and proteins. These enzymes ensure the fidelity of transfer of genetic information from the DNA to the protein. They are responsible for attaching amino acid residues to their cognate tRNA molecules by virtue of matching the nucleotide triplet, which is the first step in the protein synthesis. The translation of genetic code into protein sequence is mediated by tRNA, which accurately picks up the cognate amino acids. The attachment of the cognate amino acid to tRNA is catalyzed by aaRSs, which have binding sites for the anticodon region of tRNA and for the amino acid to be attached. The two binding sites are separated by ≈ 76 Å and experiments have shown that the communication does not go through tRNA (Gale et al., 1996). The problem addressed here is how the information of binding of tRNA anticodon near the anticodon binding site is communicated to the active site through the protein structure. These enzymes are modular with distinct domains on which extensive kinetic and mutational experiments and supported by structural data are available, highlighting the role of inter-domain communication (Alexander and Schimmel, 2001). Hence these proteins present themselves as excellent systems for in-silico studies. Various methods involved for the construction of protein structure networks are well established and analyzed in a variety of ways to gain insights into different aspects of protein structure, stability and function (Kannan and Vishveshwara, 1999; Brinda and Vishveshwara, 2005). In the present study, we have incorporated network parameters for the analysis of molecular dynamics (MD) simulation data, representing the global dynamic behavior of protein in a more elegant way. MD simulations have been performed on the available (and modeled) structures of aaRSs bound to a variety of ligands, and the protein structure networks (PSN) of non-covalent interactions have been characterized in dynamical equilibrium. The changes in the structure networks are used to understand the mode of communication, and the paths between the two sites of interest identified by the analysis of the shortest path. The allosteric concept has played a key role in understanding the biological functions of aaRSs. The rigidity/plasticity and the conformational population are the two important ideas invoked in explaining the allosteric effect. We have explored the conformational changes in the complexes of aaRSs through novel parameters such as cliques and communities (Palla et al., 2005), which identify the rigid regions in the protein structure networks (PSNs) constructed from the non-covalent interactions of amino acid side chains. The thesis consists of 7 chapters. The first chapter constitutes the survey of the literature and also provides suitable background for this study. The aims of the thesis are presented in this chapter. Chapter 2 describes various techniques employed and the new techniques developed for the analysis of PSNs. It includes a brief description of well -known methods of molecular dynamics simulations, essential dynamics, and cross correlation maps. The method used for the construction of graphs and networks is also described in detail. The incorporation of network parameters for the analysis of MD simulation data are done for the first time and has been applied on a well studied protein lysozyme, as described in chapter 3. Chapter 3 focuses on the dynamical behavior of protein structure networks, examined by considering the example of T4-lysozyme. The equilibrium dynamics and the process of unfolding are followed by simulating the protein with explicit water molecules at 300K and at higher temperatures (400K, 500K) respectively. Three simulations of 10ns duration have been performed at 500K to ensure the validity of the results. The snapshots of the protein structure from the simulations are represented as Protein Structure Networks (PSN) of non-covalent interactions. The strength of the non-covalent interaction is evaluated and used as an important criterion in the construction of edges. The profiles of the network parameters such as the degree distribution and the size of the largest cluster (giant component) have been examined as a function of interaction strength (Ghosh et al., 2007). We observe a critical strength of interaction (Icritical) at which there is a transition in the size of the largest cluster. Although the transition profiles at all temperatures show behavior similar to those found in the crystal structures, the 500K simulations show that the non-native structures have lower Icritical values. Based on the interactions evaluated at Icritical value, the folding/unfolding transition region has been identified from the 500K simulation trajectories. Furthermore, the residues in the largest cluster obtained at interaction strength higher than Icritical have been identified to be important for folding. Thus, the compositions of the top largest clusters in the 500K simulations have been monitored to understand the dynamical processes such as folding/unfolding and domain formation/disruption. The results correlate well with experimental findings. In addition, the highly connected residues in the network have been identified from the 300K and 400K simulations and have been correlated with the protein stability as determined from mutation experiments. Based on these analyses, certain residues, on which experimental data is not available, have been predicted to be important for the folding and the stability of the protein. The method can also be employed as a valuable tool in the analysis of MD simulation data, since it captures the details at a global level, which may elude conventional pair-wise interaction analysis. After standardizing the concept of dynamical network analysis using Lysozyme, it was applied to our system of interest, the aaRSs. The investigations carried out on Methionyl-tRNA synthetases (MetRS) are presented in chapter 4. This chapter is divided into three parts: Chapter 4A deals with the introduction to aminoacyl tRNA synthetases (aaRS). Classification and functional insights of aaRSs obtained through various studies are presented. Chapter 4B is again divided into parts: BI and BII. Chapter 4BI elucidates a new technique developed for finding communication pathways essential for proper functioning of aaRS. The enzymes of the family of tRNA synthetases perform their functions with high precision, by synchronously recognizing the anticodon region and the amino acylation region, which is separated by about 70Å in space. This precision in function is brought about by establishing good communication paths between the two regions. We have modelled the structure of E.coli Methionyl tRNA synthetase, which is complexed with tRNA and activated methionine. Molecular dynamics simulations have been performed on the modeled structure to obtain the equilibrated structure of the complex and the cross correlations between the residues in MetRS. Furthermore, the network analysis on these structures has been carried out to elucidate the paths of communication between the aminoacyl activation site and the anticodon recognition site (Ghosh and Vishveshwara, 2007). This study has provided the detailed paths of communication, which are consistent with experimental results. A similar study on the (MetRS + activated methionine) and (MetRS+tRNA) complexes along with ligand free-native enzyme has also been carried out. A comparison of the paths derived from the four simulations has clearly shown that the communication path is strongly correlated and unique to the enzyme complex, which is bound to both the tRNA and the activated methionine. The method developed here could also be utilized to investigate any protein system where the function takes place through long distance communication. The details of the method of our investigation and the biological implications of the results are presented in this chapter. In chapter 4BII, we have explored the conformational changes in the complexes of E.coli Methionyl tRNA synthetase (MetRS) through novel parameters such as cliques and communities, which identify the rigid regions in the protein structure networks (PSNs). The rigidity/plasticity and the conformational population are the two important ideas invoked in explaining the allosteric effect. MetRS belongs to the aminoacyl tRNA Synthetases (aaRSs) family that play a crucial role in initiating the protein synthesis process. The network parameters evaluated here on the conformational ensembles of MetRS complexes, generated from molecular dynamics simulations, have enabled us to understand the inter-domain communication in detail. Additionally, the characterization of conformational changes in terms of cliques/communities has also become possible, which had eluded conventional analyses. Furthermore, we find that most of the residues participating in clique/communities are strikingly different from those that take part in long-range communication. The cliques/communities evaluated here for the first time on PSNs have beautifully captured the local geometries in their detail within the framework of global topology. Here the allosteric effect is revealed at the residue level by identifying the important residues specific for structural rigidity and functional flexibility in MetRS. Chapter 4C focuses on MD simulations of Methionyl tRNA synthetase (AmetRS) from a thermophilic bacterium, Aquifex aeolicus. As describe in Chapter 4B, we have explored the communication pathways between the anticodon binding region and the aminoacylation site, and the conformational changes in the complexes through cliques and communities. The two MetRSs from E.coli and Aquifex aeolicus are structurally and sequentially very close to each other. But the communication pathways between anticodon binding region and the aminoacylation site from A. aeolicus have differed significantly with the communication paths obtained from E.coli. The residue composition and cliques/communities structure participating in communication are not similar in the MetRSs of both these organisms. Furthermore the formation of cliques/communities and hubs in the communication paths are more in A. aeolicus compared to E.coli. The participation of structurally homologous linker peptide, essential for orienting the two domains for efficient communication is same in both the organisms although, the residues composition near domain interface regions including the linker peptide is different. Thus, the diversity in the functioning of two different MetRS has been brought out, by comparing the E.coli and Aquifex aeolicus systems. Protein Structure network analysis of MD simulated trajectories of various ligand bound complexes of Escherichia coli Cysteinyl-tRNA synthetase (CysRS) have been discussed in Chapter 5. The modeling of the complex is done by docking the ligand CysAMP into the tRNA bound structure of E.coli Cysteinyl tRNA synthetase. Molecular dynamics simulations have been performed on the modeled structure and the paths of communications were evaluated using a similar method as used in finding communication paths for MetRS enzymes. Compared to MetRS the evaluation of communication paths in CysRS is complicated due to presence of both direct and indirect readouts. The direct and indirect readouts (DR/IR) involve interaction of protein residues with base-specific functional group and sugar-phosphate backbone of nucleic acids respectively. Two paths of communication between the anticodon region and the activation site has been identified by combining the cross correlation information with the protein structure network constructed on the basis of non-covalent interaction. The complete paths include DR/IR interactions with tRNA. Cliques/communities of non-covalently interacting residues imparting structural rigidity are present along the paths. The reduction of cooperative fluctuation due to the presence of community is compensated by IR/DR interaction and thus plays a crucial role in communication of CysRS. Chapter 6 focuses on free energy calculations of aminoacyl tRNA synthetases with various ligands. The free energy contributions to the binding of the substrates are calculated using a method called MM-PBSA (Massova and Kollman, 2000). The binding free energies were calculated as the difference between the free energy of the enzyme-ligand complex, and the free ligand and protein. The ligand unbinding energy values obtained from the umbrella sampling MD correlates well with the ligand binding energies obtained from MM-PBSA method. Furthermore the essential dynamics was captured from MD simulations trajectories performed on E.coli MetRS, A. aeolius MetRS and E.coli CysRS in terms of the eigenvalues. The top two modes account for more than 50% of the motion in essential space for systems E.coli MetRS, A. aeolius MetRS and E.coli CysRS. Population distribution of protein conformation states are looked at the essential plane defined by the two principal components with highest eigenvalues. This shows how aaRSs existed as a population of conformational states and the variation with the addition of ligands. The population of conformational states is converted into Free energy contour surface. From free energy surfaces, it is evident that the E.coli tRNAMet bound MetRS conformational fluctuations are more, which attributes to less rigidity in the complex. Whereas E.coli tRNACys bound CysRS conformational fluctuations are less and this is reflected in the increase in rigidity of the complex as confirmed by its entropic contribution. Future directions have been discussed in the final chapter (Chapter 7). Specifically, it deals with the ab-initio QM/MM study of the enzymatic reaction involved in the active site of E.coli Methionyl tRNA synthetase. To achieve this, two softwares are integrated: the Quantum Mechanics (QM) part includes small ligands and the Molecular Mechanics (MM) part as protein MetRS are handled using CPMD and Gromacs respectively. The inputs for two reactions pathways are prepared. First reaction involves cyclization reaction of homocysteine in the active site of MetRS and the second reaction deals with charging of methionine in the presence of ATP and magnesium ion. These simulations require very high power computing systems and also time of computation is also very large. With the available computational power we could simulate up to 10ps and it is insufficient for analysis. The future direction will involve the simulations of these systems for longer time, followed by the analysis for reaction pathways.