Thèses sur le sujet « AARS2 »

My project has been focused on the identification and validation of new mitochondrial gene variants and new mitochondria-related genes. The first report is about a woman presenting with a stroke-like episode and an history of severe hearing loss, frequent migraines, exercise intolerance, myalgias and limb-girdle muscle weakness indicating a slowly progressive myopathy and secondary amenorrhea with low gonadotropin levels. A muscle biopsy showed ragged-red, cytochrome c oxidase-negative fibers, and an isolated defect of cytochrome c oxidase activity in muscle mitochondria and sequence analysis of muscle mtDNA revealed a new heteroplasmic m.6597C>A transversion in the MTCOI gene, encoding subunit I of cytochrome c oxidase. Analysis on transmitochondrial cybrids demonstrated that the mutation is indeed associated with COX deficiency, i.e. pathogenic. The second report is about a new phenotype associated to mutations in the AARS2 gene encoding for the mitochondrial aminoacyl tRNA synthetase, identified in six patients presenting with primary ovarian failure, cerebellar and pyramidal signs and cognitive or behavioural disturbances. Two patients underlined a muscle biopsy which showed a severe complex IV defect at histochemical and biochemical analyses. The third report is about the clinical and biochemical phenotypes associated with mutations in two new mitochondrial aminoacyl tRNA synthetases (ARSs2) genes. In the first patient, an 8 years old child presenting with psychomotor delay, seizures, facial dysmorphisms and hyperlactacidemia and a brain MRI showing hyperintense lesions in the insula and fronto-temporal right cortex, whole exome sequencing (WES) identified a homozygous missense mutation in VARS2, encoding the mitochondrial valyl tRNA-synthetase. In two siblings presenting with a phenotype characterized by hypotonia and psychomotor retardation, high plasma and liquor lactate, both died at few months of age WES revealed two variants in TARS2, encoding the mitochondrial threonyl tRNA-synthetase: a missense and a splice site mutation. VARS2 and TARS2 mutations segregate within patients families. Patients’ clinical- biochemical phenotype and in silico and in vitro analyses of VARS2 and TARS2 mutations clearly indicate these genes as disease-causative. Expression of the corresponding wild-type enzymes led to recovery of the biochemical impairment of mitochondrial respiration in immortalized mutant fibroblasts; yeast modelling of the VARS2 mutation confirmed its pathogenic role.

The aminoacyl-tRNA synthetases (aaRSs) are the universal set of enzymes responsible for attaching amino acids to tRNA to be used as substrates in the process of protein translation. As these enzymes act at the transition between nucleic acids and proteins, their specificity of action is critical for maintaining the fidelity of the genetic code. From a mechanistic standpoint, aaRS specificity is enforced by a complex series of tRNA structural and chemical elements that collectively make up its identity set and serve to distinguish one tRNA from another. Based on sequence, structure, and oligomeric differences, the aaRS family has been partitioned into two classes, each of which is responsible for roughly half of the 22 genetically encoded amino acids. In the studies presented here, pre-steady-state kinetic methods were employed to measure individual events that collectively make up the catalytic cycle of the class II Escherichia coli Histidyl-tRNA Synthetase (HisRS) in order to elucidate the nature of its enzymatic activity and determine how these events contribute to the exquisite specificity between enzyme and tRNA. The results presented here indicate indentiy elements of tRNAHis regulate the activity of the amino acid activation and aminoacyl transfer half reactions. Additional evidence suggests communication between active sites of the HisRS homodimer plays a role in establishing an alternating cycle of catalysis in the steady state.

An After Action Review (AAR) is the Army training system's performance feedback mechanism. The purpose of the AAR is to improve team (unit) and individual performance in order to increase organizational readiness. While a large body of knowledge exists that discusses instructional strategies, feedback and training systems, neither the AAR process nor the AAR systems have been examined in terms of learning effectiveness and efficiency for embedded trainers as part of a holistic training system. In this thesis, different feedback methods for embedded training are evaluated based on the timing and type of feedback used during and after training exercises. Those feedback methodologies include: providing Immediate Directive Feedback (IDF) only, the IDF Only feedback condition group; using Immediate Direct Feedback and delayed feedback with open ended prompts to elicit self-elaboration during the AAR, the IDF with AAR feedback condition group; and delaying feedback using opened ended prompts without any IDF, the AAR Only feedback condition group. The results of the experiment support the hypothesis that feedback timing and type do effect skill acquisition, retention and transfer in different ways. Immediate directive feedback has a significant effect in reducing the number of errors committed while acquiring new procedural skills during training. Delayed feedback, in the form of an AAR, has a significant effect on the acquisition, retention and transfer of higher order conceptual knowledge as well as procedural knowledge about a task. The combination of Immediate Directive Feedback with an After Action Review demonstrated the greatest degree of transfer on a transfer task.
M.S.
Department of Industrial Engineering and Management Systems
Engineering and Computer Science
Industrial Engineering and Management Systems

The aim of this Thesis was to improve our understanding and assessment of feeding conditions for zooplanktivorous fish in the Baltic Sea. We investigated (papers I, II) the usefulness of biochemical proxies for assessments of growth and metabolic rates in the dominant Baltic copepod Acartia bifilosa. A predictive model (paper I) for egg production rate (EPR), based on body size, RNA content, and water temperature, was established using females of different geographical origin. This model demonstrates the usefulness of RNA content as a proxy for growth in zooplankton and, together with abundance data, it could be used to evaluate fish feeding conditions. Further (paper II), using A. bifilosa exposed to a food gradient, we evaluated responses of physiological rates and other biochemical proxies for growth and established correlations between physiological and biochemical variables. EPR and ingestion rate were most significantly correlated with RNA content. As assayed variables saturated at different food concentrations, food availability may affect assessments of physiological rates using proxies. In paper III, we explored the effect of high EPR and ingestion rate on astaxanthin content in A. bifilosa. We found that the astaxanthin content decreased at high feeding rates, most likely due to decreased assimilation efficiency. This may impact the quality of zooplankton as prey. The invasion of Cercopagis pengoi, a zooplanktivorous cladoceran, has altered the trophic linkages in the Baltic Sea food web. In paper IV, we evaluated the feeding of zooplanktivorous fish on C. pengoi and found that irrespective of size both herring and sprat feed on it, with large herring being more selective. In turn, C. pengoi feeds mainly on older copepods (paper V), which are acknowledged important in fish nutrition. These results indicate that C. pengoi may compete with fish due to the diet overlap.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In progress. Paper 3: Submitted

Les aminoacyl-ARNt synthétases mitochondriales humaines (aaRS mt) sont des enzymes clés de la traduction mitochondriale. Elles catalysent l'aminoacylation des ARNt par les acides aminés correspondent. Des mutations dans leurs gènes sont corrélées à des pathologies avec un large spectre de phénotypes cliniques, mais aux mécanismes moléculaires sous-jacents encore incompris. L'objectif de ce travail de thèse s'intègre dans les axes scientifiques du laboratoire, mais élargit l'intérêt et les connaissance à un système encore peu exploré: l'arginyl-ARNt synthétase mitochondriale (ArgRS mt). Des mutations dans la ArgRS sont liées à une hypoplasie Pontocérébelleuse (PCH6), une pathologie neurodéveloppementale sévère. Le travail de cette thèse s’articule autour de 3 axes : (I) L’analyse des phénotypes cliniques des pathologies liées aux mutations dans les aaRS mt, (II) La caractérisation des propriétéscellulaires de l’ArgRS mt, et (III) L'étude de l’impact de mutations « pathologiques » sur diverses propriétés de l’ArgRS mt. Combinés avec les travaux précédents, les résultats obtenus sont une contribution importante à l'élargissement des connaissances fondamentales des mt aaRSs, et apportent un nouvel éclairage sur le lien entre les mt-aaRSs-mutations et la maladie
Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are housekeeping enzymes involved in the mitochondrial translation. They catalyze the aminoacylation of tRNAs with their cognate amino acids. Mutations in their nuclear genes are correlated with pathologies with a broad spectrum of clinical phenotypes, but with so far no clear explanations about the underlying molecular mechanism(s). The aim of this PhD work follows the long-standing efforts of the host laboratory but expand the interest and knowledge to an unexplored system: the human mitochondrial arginyl-tRNA synthetase (mt-ArgRS). Mutations in the mt-ArgRS lead to Pontocebellar hypoplasia type 6, a severe neuro-developmental pathology. I thus contributed to i) comprehensively analyze the clinical data reported in pathologies related to mutations on mt-aaRSs, resulting in a categorization according to the affected anatomical system; ii) decipher some cellular properties of the mt-ArgRS; and iii) investigate to impact of disease-associated mutations on mt-aaRSs properties. Combined with previous works, the present results expand the knowledge of the mt-aaRSs, shedding new light on the link between mt-aaRSs-mutations and disease

The Hereditary ataxias (HAs) are a group of heterogenous neurological disorders associated with multiple genetic etiologies and encompassing a wide spectrum of phenotypes, where ataxia is the prominent feature. HAs are characterized by degeneration of Purkinje cell and/or spinocerebellar connections, often associated with defects in additional brain structures, and all patterns of inheritance may occur. Similar to other fields of medical genetics, Next Generation Sequencing (NGS) has entered the HA scenario widening our genetic and clinical knowledge of this condition, but routine NGS applications still miss genetic diagnosis in about two third of patients. In this doctoral study, we applied multi-gene panels to define the molecular basis in 259 patients with a clinical diagnosis of HA and negative to tests for pathological expansion in SCA1, 2, 3, 6, 7, 8, 12, 17 and FXN. We found a positive molecular diagnosis in 25% of patients, whereas a similar number of patients had an uncertain diagnosis due to the presence of either variants of uncertain significance or lack of biological samples to determine segregation among family members. Hence despite a higher positive diagnostic rate compared to similar studies described in literature, a half of patients lacked any indication of the genetic cause of their disease. Using exome sequencing as a second-tier approach in some families, refractory to multi-gene panel analysis, did not significantly improved our diagnostic yield. On the other hand, NGS analysis in our cohort indicated that familial cases were more easily diagnosed rather than sporadic cases, and also that combining massive sequencing with detailed clinical information and family studies increases the likelihood to reach a molecular diagnosis. Among positive patients, we could expand clinical and allelic information in a subgroup of genes offering original description of new mutations and corroborating genetic findings with functional investigations that took advantage of different in vitro or in vivo platforms. In particular, through functional studies in SPG7 knock-down models of Drosophila melanogaster, we remarked that SPG7, whose mutations cause spastic paraplegia type 7, has a critical role in neurons more than in skeletal muscle. The high frequency of p.Ala510Val mutation in SPG7 observed in our cohort as well in similar studies performed elsewhere moved us to develop a humanized knock-in fruit fly model harboring that specific mutation and prepare preliminary characterizations. Similar studies in fruit fly were performed silencing AFG3L2, the gene causing SPAX5 in a child in association with an unusual, relatively milder phenotype. Furthermore, combination of skin fibroblasts and Saccharomyces cerevisiae as models was employed in the genetic characterization of new mutations in a novel recessive HARS-related phenotype whereas primary human cells, yeast and Danio rerio models were used to functionally characterize new HA-related mutations in COQ4. Finally, we could expand the clinical presentation of rare causes of HAs describing new dominant mutations in STUB1 and biallelic variants in RFN216, COQ8A, and ATP13A2. Altogether, studies performed during this doctoral work further underlined the usefulness of NGS in HAs and highlighted how NGS technologies rely on the integrated use of family and clinical studies and different in vitro/in vivo platforms to substantiate molecular findings. The latter platform will be also a tool for future investigations to dissect pathogenesis and to improve therapies.

博士
國立中央大學
生命科學系
104
Previous studies showed that cytoplasmic methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRSc) form a ternary complex with an aaRS cofactor, Arc1p, thereby enhancing their aminoacylation activities. In addition, Arc1p also regulates the subcellular distribution of these two associated enzymes. Upon dissociation from the ternary complex, GluRSc and MetRS are targeted into the mitochondria and nucleus for functioning. The structure of Arc1p can be divided into three domains, N, M, and C domains. The N domain interacts with GluRSc and MetRS, while the M plus C domains form a non-specific tRNA-binding domain. A recent report demonstrated that a SSKD motif in the N domain of Arc1p can be biotinylated through post-translational modification in vivo. However, the biological significance of this modification remained unclear. We show herein that Arc1p was biotinylated (15%) under normal growth conditions. However, biotinylation had little effect on its ability to interact with tRNA or GluRSc/MetRS. In contrast, Arc1p was almost biotin free at a high temperature. Non-biotinylated Arc1p was more heat-tolerant and more efficiently promoted the aminoacylation activity of GluRSc. Perhaps the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes. WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases (aaRSs) and play roles in tRNA or protein binding. We show herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. As a result, the cytoplasmic form possessed an N-terminal WHEP domain, while its mitochondrial counterpart possessed an extra N-terminal sequence (aa 1~64) consisting of a mitochondrial targeting signal (MTS； aa 1~20) and an appended domain (aa 21~64). Cross-species rescue assays showed that this dual-functional gene effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 (which encodes GlyRS) knockout strain. While both forms of CeGlyRS efficiently charged the cytoplasmic tRNAsGly of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNAGly isoacceptor, which carries a defective TψC hairpin. Despite the WHEP domain per se lacking tRNA-binding activity, deletion of this domain reduced the enzyme’s catalytic efficiency. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests the WHEP domain may act in cis to regulate the enzyme’s dynamic structure and activity.

Candida albicans is the main human fungal pathogen. It is usually commensal yet when immunocompromised individuals are exposed to it infections normally develop from mild rashes to systemic disease. Candida albicans is characterized by the reassignment of the CUG codon from Leucine to Serine by a hybrid serine tRNA (tRNACAGSer) which decodes the leucine-CUG as leucine (3 to 5 %) and as serine (95 to 97 %) under normal growth conditions. The tRNACAGSer is aminoacylated by two aminoacyl tRNA synthetases, the leucyl-tRNA synthetase (LeuRS) and the seryl-tRNA synthetase (SerRS). Previous studies showed that when Candida albicans is exposed to stress, namely temperature, pH, osmolarity and antifungals, the level of leucine misincorporation rises, suggesting that C. albicans regulates mistranslation levels in response to stress. In this thesis we started characterizing the mechanisms that controls Leu misincorporation in C. albicans. For this, C. albicans strains harboring deletions in genes of selected kinases and transcription factors were transformed with fluorescent reporter systems to monitor the levels of leucine and serine incorporation at CUG codons. The activity of the LeuRS (CDC60) and SerRS (SES1) promoter was quantified in several different physiological conditions using a second fluorescent reporter system. The results suggested that Leu misincorporation at CUG codons could be due to increased LeuRS expression or decreased SerRS expression. In the second part of this study, protein from C. albicans KO strain collection was extracted and the levels of LeuRS and SerRS were quantified by western blot using antibodies against both enzymes. LeuRS/SerRS expression ratio in the mutant relative to WT strains allowed the identification of 3 putative transcription factors that regulate the expression of LeuRS and SerRS, namely EFG1, MRR1 and ACE2
Candida albicans é o fungo patogénico mais comum em humanos. Este fungo normalmente é um comensal, no entanto quando indivíduos imunodeprimidos são expostos a ele desenvolvem normalmente infeções desde irritações de pele a doença sistémica generalizada. Candida albicans é caracterizada pela reatribuição do codão CUG de Leucina para Serina por um tRNA híbrido de serina (tRNACAGSer) que em condições normais descodifica o CUG-leucina como leucina (3 a 5%) e como serina (93 a 95%). O tRNACAGSer é aminoacilado por duas aminoacil tRNA sintetases, a leucil-tRNA sintetase (LeuRS) e a seril-tRNA sintetase (SerRS). Estudos anteriores mostraram que quando Candida albicans é exposta ao stress, nomeadamente temperatura, pH, osmolaridade e antifúngicos, o nível de mistranslation de leucina aumenta, sugerindo que C. albicans regula os níveis de mistranslation in resposta ao stress. Nesta tese começamos por caracterizar mecanismos que controlam a misincorporation de leucina em C. albicans. Para isto, transformamos estirpes de C. albicans que contêm deleções de genes de cinases selecionadas e de fatores de transcrição com sistemas repórter fluorescentes para monitorizar os níveis de incorporação de leucina e serina no codão CUG. A atividade dos promotores LeuRS (CDC60) e da SerRS (SES1) foi quantificada em várias condições fisiológicas diferentes utilizando um segundo sistema repórter florescente. Os resultados sugerem que a misincorporation de leucina nos codões CUG pode ser devido ao aumento da expressão de LeuRS ou a um decréscimo da expressão de SerRS. Na segunda parte do estudo, proteínas da coleção de estirpes KO de C. albicans foi extraída e os níveis de LeuRS e SerRS foram quantificadas por western blot utilizando anticorpos para ambas as enzimas. O rácio da expressão de LeuRS/SerRS nos mutantes em relação a estirpe selvagem permitiu a identificação de 3 fatores de transcrição putativos que regulam a expressão de LeuRS e SerRS, nomeadamente EFG1, MRR1 e ACE2
Mestrado em Biomedicina Molecular

碩士
東吳大學
企業管理學系
91
For the more and more severe competition in world’s business environment, Corporations pay much attention to their learning abilities. AARs(After Action Reviews) which was mentioned many times in Peter Senge et al’s book “The Dance of Change” has been one of the new growing developments. Though the AARs infrastructure has high potential for facilitating the creation and management of knowledge, it’s lack of strong evidences and basis of practice and theories, in particular the experiences of business who used this tool are rarely seen in bibliographies until now. Thus, we tried to extend the methodology and record a practical case of AARs, then established an AARs’ causal model and intervention model. We adapted the methodology of Participative Action Research, derived a middle-range intervention model in our case. After that, we removed the contextual variables of our case, derived “AARs’ two-stage intervention model”. We also derived “AARs’ causal model” through the probing of it’s bibliographies. In conclusion, we explained how AARs could applied to Social Capital, Communities of Practice, Knowledge Sharing, and Organizational Learning.

Biological processes are so complicated that to understand the mechanisms underlying the functioning of biomolecules it is inevitable to study them from various perspectives and with a wide range of tools. Understanding the function at the molecular level obviously requires the knowledge of the three dimensional structure of the biomolecules. Experimentally this can be obtained by techniques such as X‐ray crystallography and NMR studies. Computational biology has also played an important role in elucidating the structure function relationship in biomolecules. Computationally one can obtain the temporal as well as ensemble behavior of biomolecules at atomic level under conditions that are experimentally not accessible. Molecular dynamics(MD) study is a technique that can be used to obtain information of the dynamic behavior of the biomolecules. Dynamics of large systems like proteins can be investigated by classical force fields. However, the changes at the level of covalent bond involve the reorganization of electron density distribution which can be addressed only at Quantum mechanical level. In the present thesis, some of the biological systems have been characterized both at the classical and quantum mechanical level. The systems investigated by MD simulations and the insights brought from these studies are presented in Chapters 3 and 4. The unusual bonds such as pyrophosphate linkage in ATP and short strong hydrogen bonds in proteins, investigated through high level quantum chemical methods, are presented in Chapters 5, 6 and 7. Part of this thesis is aimed to address some important issues related to the dynamics of Tryptophanyl tRNA synthetase (TrpRS) which belongs to classic of aminoacyl‐tRNA synthetases (aaRS). aaRSs are extremely important class of enzymes involved in the translation of genetic code. These enzymes catalyze the aminoacylation of tRNAs to relate the cognate amino acids to the anticodon trinucleotide sequences. aaRSs are modular enzymes with distinct domains on which extensive kinetic and mutational experiments as well as structural analyses have been carried out, highlighting the role of inter‐domain communication (Alexander and Schimmel, 2001). The overall architecture of tRNA synthetases consists of primarily two domains. The active site domain is responsible for the activation of an amino acid with ATP in synthesizing an enzyme‐bound aminoacyl‐adenylate, and transfer of the aminoacyl‐adenylate intermediate to the 3’end of tRNA. The second domain is responsible for selection and binding of the cognate tRNA. aaRSs are allosteric proteins in which the binding of tRNA at the anticodon domain influences the activity at the catalytic region. These two binding sites are separated by a large distance. One of the aims of this thesis is to characterize such long distance communication (allosteric communication) at atomic level in Tryptophanyl tRNA synthetase. This is achieved by generating ensembles of conformations by MD simulations and analyzing the trajectories by novel graph theoretic approach. 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). The parameters such as clusters, hubs and shortest paths provide valuable information on the structure and dynamics of the proteins. In this thesis, network parameters are used for the analysis of molecular dynamics MD) simulation data, to represent the global dynamic behavior of protein in a more elegant way. MD simulations are performed on some available (and modeled) structures of TrpRS bound to a variety of ligands, and the protein structure networks( PSN) of non‐covalent interactions are characterized in dynamical equilibrium. The ligand induced conformational changes are investigated through structure networks. These networks are used to understand the mode of communication between the anticodon domain and the active site. The interface dynamics is crucial for the function of TrpRS (since it is a functional dimer) and it is investigated through interface clusters. The matter embodied in the thesis is presented as 9 chapters. Chapter 1 lays the suitable background and foundation for the study, surveying relevant literature from different fields .Chapter 2 describes in detail the various materials, methods and techniques employed in the different analyses and studies presented in this thesis. A brief description of well‐known methods of molecular dynamics simulations, essential dynamics calculations, cross correlation maps, conformational clustering etc.is presented. The methods for constructing protein structure graphs and networks, developed in our lab, are described in detail. The use of network parameters for the analysis of MD simulation data to address the problem of communication between the two distal sites is also presented. Some descriptions of the ab initio quantum mechanical methods, which are used to investigate the unusual bonds in biomolecules, are also presented in this chapter. Chapter 3 is devoted in discussing the results from several normal as well as high temperature MD simulations of ligand‐free and ligand bound Bacillus stearothermophilus Tryptophanyl‐tRNA synthetase (bsTrpRS). The essential modes of the protein in the presence of different ligands are captured by essential dynamics calculations. Different conformations of the protein associated with the catalysis process of TrpRS, as captured through experiments, are discussed in the context of conformational sampling. High temperature simulations are carried out to explore the larger conformational space. Chapter 4 is focused on the results obtained from the MD simulation of human Tryptophanyl‐tRNA synthetase (hTrpRS). The structure of human TrpRS bound to the activated ligand (TrpAMP) and the cognate tRNA(tRNATRP) is modeled since no structure in the presence of both TrpAMP and tRNATRP is available. MD simulations on these modeled as well as other complexes of hTrpRS are performed to capture the dynamical process of ligand induced conformational changes (Hansiaetal., communicated). Both the local and the global changes in the protein conformation from the protein structure network (PSN) of MD snapshots are analyzed. Several important information such as the ligand induced correlation between different residues of the protein, asymmetric binding of the ligands to the two subunits of the protein, and the path of communication between the anticodon region and the aminoacylation site are obtained. Also, the role of the dimmer interface, from a dynamic perspective, is obtained for the first time. The interface dynamics which stabilize different quaternary structures of lectins (with high sequence and structure similarity) were investigated in a collaborative work (Hansiaetal.,2007). The lectin peanut agglutinin (PNA) is a tetramer with three different types of interfaces. The interface dynamics of this protein in the presence and in the absence of metal ions was investigated and the paper reporting the results from this study is included as appendix in this thesis. Chapter 5 deals with high level ab initio quantum chemical calculations on tri‐ and diphosphate fragments of adenosine triphosphate (ATP). Pyrophosphate prototypes such as methyl triphosphate and methyl diphosphate molecules in their different protonation states have been investigated at high levels of calculations (Hansiaetal., 2006a). The optimized geometries, the thermochemistry of the hydrolysis and the molecular orbitals contributing to the high energy of these compounds have been analyzed. These investigations provide insights into the‘‘highenergy’’character of ATP molecule. Further, the dependence of vibrational frequencies on the number of phosphate groups and the charged states has also been presented. These results aid in the interpretation of spectra obtained by experiments on complexes containing pyrophosphate prototypes. Hydrogen bonding is fundamental in understanding the structure and properties of molecules of biological interest including proteins. A recent analysis carried out in our lab showed that a significant number of short hydrogen bonds (SHB) are present in proteins (Rajagopal and Vishveshwara, 2005). Chapters 6 and 7 elucidate the results obtained from ab initio quantum chemical calculations on some of these SHBs to get aquantitative estimation of their geometry and strength. In chapter 6, asystematic analysis of the geometries and the energetics of possible SHB systems, which are frequently encountered in proteins, are presented at different levels of theory (HF,DFTandMP2). It is found that the SHBs involving both charged residues in the proteins are intrinsic in nature. However, two neutral residues form a SHB in the protein crystal structures either due to geometric constraints or due to the environment of these residues. This analysis enables one to distinguish SHBs which are formed because of geometric constraints from those which are formed because of the inherent property of the chemical groups involved in the hydrogen bonding. These results are useful in refining protein structures determined by crystallographic or NMR methods. In addition, sulfur atom of methionine and cysteinein proteins also participate in SHBs, which are not so well characterized. Chapter 7 presents the similar analysis carried out on short hydrogen bonds in proteins involving sulfur atom. A detailed analysis of SHBs of sulfur containing groups in a data set of proteins has been carried out. Some of the residue pairs from this analysis were considered for ab initio calculations. However, the optimization of these examples resulted in breaking of the hydrogen bonds involving sulfur atoms and formation of new hydrogen bonds with oxygen and/or nitrogen atoms. Hence model systems, which mimic the real examples, were designed to carry out ab initio studies and to investigate the short hydrogen bonds involving sulfur atoms. Another study on the protein‐water interaction, which does not fall under the realm of the main objective of the thesis, is discussed in Chapter 8. Protein–water interaction is crucial for accomplishing many biological functions of proteins. In the recent past, natural probe tryptophan, located at the protein surfaces, has been extensively investigated using femtosecond spectroscopy experiments to understand salvation dynamics (Peonetal.,2002). In this chapter a method is described to follow up the molecular events of the protein–water interactions in detail. Tryptophan–water interaction in the protein Monellin is investigated in order to get the atomic level insights into the hydration dynamics, by carrying out MD simulations on Monellin (Hansiaetal.,2006b). The results are compared with those obtained from femtosecond resolved fluorescence spectroscopy. The time constants of the survival correlation function match well with the reported experimental values.This validates the procedure, adapted here for Monellin, to investigate the hydration dynamics in general. The last chapter (Chapter9) summarizes the results obtained from various studies and discusses the future directions. First part of this thesis aims to present the analysis by carrying out MD simulations on monomeric and dimeric TrpRS protein in order to understand the two steps of the aminoacylation reaction: activation of the aminoacid Trp in the first step and the transfer of the activated amino acid in the next step. In the second part, quantitative estimation of the geometry and the strength of pyrophosphate bond and short hydrogen bonds in proteins are reported in detail by subjecting the systems to high levels of quantum mechanical calculations(QM). The use of ab initio QM/MM calculations by combining the quantum mechanics(QM) with the molecular mechanics(MM) in order to study the enzymatic reactions is discussed as the future

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.

Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.