Près de 40% des protéines présentes dans les cellules sont prédites partiellement ou complètement désordonnées. Ces protéines dépourvues de structure tridimensionnelle à l'état natif sont impliquées dans de nombreux mécanismes biologiques, la flexibilité jouant un rôle moteur dans les mécanismes de reconnaissance moléculaire. La prise en considération de l'existence de flexibilité au sein des protéines et des interactions protéines-protéines a nécessité le renouvellement de nos connaissances, de notre appréhension des fonctions biologiques ainsi que des approches pour étudier et interpréter ces phénomènes. La méthode retenue pour étudier ces transitions conformationnelles est la spectroscopie par résonance magnétique nucléaire. Elle dispose d'une sensibilité unique, d'une résolution à l'échelle atomique et permet par diverses expériences d'accéder à l'ensemble des échelles de temps définissant les mouvements de ces protéines. Nous combinons ces mesures expérimentales à un modèle statistique représentant l'ensemble du paysage énergétique des protéines désordonnées : la description par ensemble explicite de structures. Ce modèle est une représentation discrète des différents états échantillonnés par ces protéines. Il permet, combinant les déplacements chimiques, les couplages dipolaires et la relaxation paramagnétique, de développer une description moléculaire de l'état déplié en caractérisant à la fois l'information locale et l'information à longue portée présente dans les protéines intrinsèquement désordonnées. / Around 40% of the human genome does not fold into stable three-dimensional structures but are either unfolded, or contain unfolded regions of significant length. The inherent flexibility of this class of proteins is essential for their function in a vast range of biomolecular process such as molecular recognition. In order to take into account the specificity of these interactions, it has been necessary to invent new approaches to study and interpret their behaviour. Nuclear magnetic resonance spectroscopy is a unique atomic resolution probe which is sensitive to a very large range of time scales. We combine experimental NMR data with a statistical model describing the energy landscape of unfolded state : the explicit ensemble description. This model is a discrete representation of the different states of theses proteins. Combining chemical shifts, residual dipolar couplings and paramagnetic relaxation enhancement, it is then possible to develop a molecular description of the unfolded state caracterising both the local and long-range information of intrinsically disordered proteins.

Pantothenate synthetase (PS), which catalyzes the last step in the pantothenate (vitamin B5) biosynthesis, is a dimeric enzyme present in bacteria, fungi and plants. The enzymatic properties of PS from Escherichia Coli, Mycobacterium tuberculosi, Fusarium Oxysporum, Lotus japonicus, Oryza sativum, Brassica napus and Arabidopsis thaliana have been characterized. The chemical reaction and the proposed mechanism of reaction are identical for PS, irrespective of the source. However, from an enzyme mechanistic point of view, plant PS’s are dissimilar to their bacterial counterparts, in that they exhibit “allosteric behavior”, a property that has not been observed in the bacterial enzyme. The behavior is quite remarkable when one takes into consideration the fact that plant PS’s share a high degree of sequence identity (~ 40%) with the bacterial enzymes. Even more intriguing is the structural mechanism proposed to explain the observed differences in structure between the PS’s from E.Coli and M.tb, which share a 42% sequence identity. Till date there is no structural information available on the plant PS’s and of the substrate bound conformation of E.coli PS. This thesis aims to provide an understanding on some aspects of the structure – function relationship of this physiologically important enzyme. Specifically, the solution properties of E. coli PS have been examined using high-resolution multinuclear, multidimensional NMR methods. Given the large size of the full-length protein (~ 63 KDa), the structurally distinct N and C-terminal domains were cloned and expressed as individual proteins and their properties investigated. Towards this end, the tertiary fold of the 40 kDa dimeric amino-terminal domain of E. coli pantothenate synthetase has been determined using multidimensional multinuclear nuclear magnetic resonance (NMR) methods (PDB entry 2k6c). Sequence specific resonance assignments for backbone HN, 15N, 13Cα, 13C', sidechain 13Cβ and aliphatic 13CH3 (of isoleucine, leucine and valine residues) were obtained using perdeuterated ILV-methyl protonated samples (BMRB entry 6940). Secondary structure of nPS was determined from 13C secondary chemical shifts and from short and medium range NOEs. Global fold of the 40 kDa homo-dimeric nPS has been determined using a total of 1012 NOEs, 696 dihedral angles, 260 RDCs, 155 hydrogen bonds, radius of gyration potential, non-crystallographic symmetry potential and database derived potential based upon the Ramachandran map. The calculated structures, which show that the N-terminal domain forms a homo-dimer in solution, is of high stereochemical quality as judged by the Ramachandran statistics (70% of the residues have backbone dihedral angles in the allowed region, 25.5% in the additionally allowed region, 4.0% in generously allowed region, and only 0.5% in disallowed region). Dynamics of nPS, which has rotational correlation time τc of 17.3 ns, was investigated by 15N relaxometry measurements. Results of these studies indicate that the E. coli protein exhibits dynamic nature at the dimer interface. These structural and dynamic features of the protein were found to be of interest when correlated with NMR based substrate binding studies. Interaction of homo-dimeric amino-terminal domain (nPS) of E. coli pantothenate synthetase (PS; encoded by the gene panC; E.C. 6.3.2.1) with the substrates pantoate, β-alanine, ATP and the product pantothenate has been studied using isotopically edited solution NMR methods. Addition of pantoate prior to ATP has led to the interesting observation that pantoate binds each monomer of nPS at two sites. ATP displaces a molecule of pantoate from the ATP binding site. β-alanine and pantothenate do not bind the protein under the condition studied. Binding of pantoate and ATP also manifests as changes in residual dipolar couplings (RDCs) of backbone 1H-15N pairs in nPS when compared to the free form of the protein. Structures of homo-dimeric nPS bound to two molecules of pantoate (PDB entry 2k6e) as well as to pantoate + ATP (PDB entry 2k6f) were calculated by inclusion of hydrogen bonds between the ligands and backbone 1H-15N pairs of nPS in addition to other NMR derived restraints. The ligand bound structures have been compared to the similar forms of the M. tb PS. Structure of each monomer of nPS bound to pantoate and ATP shows the substrates in a favorable orientation for the intermediate pantoyl adenylate to form. Moreover, at all stages of substrate binding the symmetry of the dimer was preserved. A single set of resonances was observed for all protein-ligand complexes implying symmetric binding with full-occupancy of the ligands bound to the protein. In an effort to understand the structural basis of the observed enzymatic properties of plant PS’s, a structural model of the Arabidopsis PS was constructed. The results of these structural and substrate binding studies strongly suggest that 1 Substrate binding to PS occurs only at the active site. 2 There are no additional substrate binding sites which could potentially participate as regulatory sites. 3 Pantoate does not bind at the dimer interface to induce the observed homotropic effects. 4 The structural results presented on the substrate bound forms of nPS have direct implication for the development of novel antibacterial and herbicidal agents. Recently a great deal of interest has been evinced on the effects of molecular crowding on protein folding / unfolding pathways. Nuclear magnetic resonance is the only method by which high resolution structural information can be obtained on partially denatured states of a protein under equilibrium condition. Recent methodological advances have enabled the determination of high resolution structures using information embedded in the residual dipolar couplings. Molecular crowding using confinement may thus reveal important details about chaperone mediated protein folding. We have attempted to develop a protocol to study the effects of molecular confinement by sequestering proteins in poly-acrylamide gels and then subjecting these protein molecules to denaturation and then characterize these states by nuclear magnetic resonance. The preliminary results of these studies are described here.

The thesis entitled "Pushing the Limits of NMR Sensitivity and Chiral Analysis: Design of New NMR Methods and Bio-molecular Tools" consists of six chapters. The research work reported in this thesis is focused on the development of novel chemical and NMR methodological approaches for enantiomeric analysis and mea- surement of residual dipolar couplings (RDCs), and the development of sensitivity enhanced slice selective NMR experiments for obtaining pure shift 1H spectra and the measurement of scalar couplings. The thesis is divided into two parts. The Part I comprises chapters 2-4, where the enantiomeric analysis is discussed, which includes newly developed chiral reagents, two new weak chiral aligning media and design of novel NMR techniques. Part II comprises chapters 5 and 6, which discusses new sensitivity enhanced slice selective NMR techniques. Chapter 1 gives a general introduction to NMR and the problems investigated in the remaining chapters of the thesis. The chapter starts with a brief discussion on the introduction, advancements and general applications of NMR, discussion is also given on the NMR approaches for enantiomeric analysis both in isotropic and anisotropic phases and the measurement of RDCs, including the benefits and limitations associated with each approach. The chapter sets the tone by discussing limitations of the existed NMR enantiomeric approaches and slice-selective techniques, and builds the bridge for the rest of the chapters by addressing these limitations. The chapter also introduces slice selective experiments, their benefits over other conventional methods and limitations. Additional introductory notes are also given on some related concepts. Part I : NMR Chiral analysis and RDCs measurements Chapter 2 discusses chiral sensing properties of RNA nucleosides and their utility as chiral derivatizing agents for the enantio-discrimination of 1o-amines using one dimensional 1H NMR. A three component protocol has been proposed for the complexation of nucleosides with amines, which is rapid, economical and provides maximum diastereomeric conversion. The chiral differentiating ability of nucleosides are examined for different amines based on the 1H NMR chemical shift differences between the diastereomers (∆δ R, S ). Enantiomeric differentiation has been observed at multiple chemically distinct proton sites. It is observed that adenosine and guanosine exhibit large chiral differentiation (∆δ R, S ) due to the presence of a purine ring. The comparison of the diastereomeric excess (de) measured by NMR with those of the gravimetrically prepared ratios are in excellent agreement with each other confirming the robustness of these RNA nucleosides in discriminating primary amines. Chapter 3 establishes the smooth connectivity with the chapter 2 by discussing the limitations of the enantiomeric discrimination using NMR in isotropic solutions. This chapter discusses two new water compatible aligning media that were developed based on self-assembling strategy of small bio-molecules. The self-assembled folic acid, and the binary mixture of 50-GMP and guanosine are introduced as two novel weak aligning media. The properties of these low ordered media have been systematically studied for their easy preparation, physical parameter dependent tunability of their degree of alignment, mesosphere sustainability over a broad range of temperature and the concentration of the ingredients, and the phase reproducibility. The applications of both these new media are demonstrated for chiral and pro-chiral discrimination and also for the measurement of RDCs. Both these liquid crystalline media could be tuned to very low degree of alignment (order parameter of the order of 10−4), which provides simple first order spectra of molecules aligned in them, the analysis provide order dependent NMR spectral parameters. The 50-GMP:guanosine orienting medium can be prepared in less than 1 hour, and has been demonstrated to be an ideal medium for the determination of RDCs that are used as restraints in the structure calculations of small molecules. Chapter 4 describes 1H NMR spectral complexity in isotropic and anisotropic phases and its consequences on enantiomeric analysis. In circumventing such problems, new NMR techniques have been developed and the spin dynamics involved in the designed sequences are discussed. The newly developed 2D 1H NMR experimental method termed as RES-TOCSY, and its applicability for resolving R and S enantiomeric or diastereomeric peaks of all the coupled proton spins in isotropic phase is discussed. The utility of the developed method is demonstrated in diverse situations, such as, for suppressing impurities peaks, resolving the severely overlapped peaks and unraveling the peaks masked due to severe line broadening when metal complexes are used as chiral auxiliaries. The advantages and limitations of the method over other methods available in the literature are discussed and the significant advantage of the present method is illustrated by spectral comparison with J-resolved experiment. The appli- cation of the method for the accurate measurement of enantiomeric excess has also been demonstrated. The chapter also introduces another NMR experimental technique developed for resolving enantiomeric peaks and complete unraveling of R and S spectra in anisotropic phase. The developed 2D NMR method is cited in the literature as CH-RES-TOCSY. In addition to spectroscopic visualization of R and S spectra, the method also yields C-H RDCs. The applicability of the new experiment has been demonstrated on a chosen example. The wide utility of the method has also been demonstrated for the assignment of symmetric cis- and trans- isomers. Part II : Sensitivity Enhancement of Slice selective NMR Experiments Chapter 5 describes applications of slice selective NMR experiments over conven tional NMR methods and their limitations as far as the sensitivity of signal detection is concerned, especially in low concentrated samples. The chapter introduces the implementation of Acceleration by Sharing Adjacent Polarization (ASAP) technique in slice selective experiments. It is convincingly demonstrated that ASAP helps in reducing inter scan relaxation delay and consequently permits acquisition of more number of scans in a given time, resulting in the gain in signal enhancement by a factor of two. The pulse sequences have been suitably designed for obtaining the pure shift 1H spectra and in G-SERF experiment for the measurement of 1H-1H couplings, both with significantly enhanced signal intensities. Chapter 6 describes new sensitivity enhanced slice selective NMR methods for mea- surement of scalar couplings. A new experiment has been developed which is named as Quick G-SERF (QG-SERF). It is a 1D NMR slice selective method developed based on real time spin manipulation technique. The method gives multiple scalar couplings of a selected spin with simplified multiplets, which is analogous to the 2D G-SERF but with considerable saving in instrument time by 1-2 orders of magnitude. The rapidness of the experiment arises due to reduced dimensionality. The spin dynamics involved in the pulse sequence and its working principle have been described. The application of the method is illustrated for the measurement of 1H-1H couplings. The sequence has been further improved to obtain the heteronuclear couplings between two abundant spins in an orchestrated manner and has been demonstrated for measurement of 1H-19F couplings. This sequence cited as HF-QG-SERF has been implemented on the molecules containing number of chemically non-equivalent fluorine atoms.

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