<p><!-- /* Font Definitions */ @font-face {font-family:SimSun; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-alt:宋体; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} @font-face {font-family:"\@SimSun"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:SimSun; mso-fareast-language:ZH-CN;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 70.85pt 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --><p>Magnetiskresonanstomografi, MRI, är en användbar teknik inom flera områden, i denna uppsats ligger fokus på användning inom medicin. Fysiken som ligger bakom MRI presenteras, som t ex uppdelning av energinivåer och kärnmagnetiskresonans. Uppbyggnad och tekniken som ligger bakom MRI har även studeras. Sedan har det gjorts jämförelse mellan MRI, röntgen och datortomografi.</p><p> </p><p>Utöver litteraturstudierna har simulering gjorts. I denna del har en modell för hjärnan byggts i programmet Comsol Multiphysics. I denna modell har studier av energiöverföringen, magnetiska fältet och elektriska fältet gjorts. Modellen har lösts både som stationärt problem och som tidsberoende. För energiöverföringen som redovisas som tidsmedelvärdet ser resultaten liknade ut för det stationära fallet som för de tidsberoende fallen, dock om man inte ser på tidsmedelvärdet utan energiöverföring under hela tiden så kan man dra slutsatsen att det stationära fallet är det mest kritiska.</p></p> / <p><!-- /* Font Definitions */ @font-face {font-family:SimSun; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-alt:宋体; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} @font-face {font-family:"\@SimSun"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:SimSun; mso-fareast-language:ZH-CN;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 70.85pt 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --><p>Magnetic resonance imaging, MRI, is a useful technical method in many different areas; in this report lies focus on uses in medicine. MRI has been studied from a physical meaning, such as nuclear magnetic resonance, NMR, and spin splitting. A technical perspective of MRI has been studied, such as how MRI is built-up and technical details. MRI has been compared with Computed Tomography, CT, and X-rays.</p><p> </p>In addition to the theoretical studies, have simulations using the programme Comsol Multiphysics been done. One model has been built up to simulate MRI influences on the brain. The energy transfer, magnetic field and electric field have been studied. The model has been solved both as stationary and as time dependent problem. In the solution can a small difference be noticed which depend on that the results show the time average. If one studies the real solution, not the time average, can one conclusion rather quickly been drawn that the stationary solution have the highest transferred energy.</p>

No description available.

<!-- /* Font Definitions */ @font-face {font-family:SimSun; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-alt:宋体; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} @font-face {font-family:"\@SimSun"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:SimSun; mso-fareast-language:ZH-CN;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 70.85pt 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} -->Magnetiskresonanstomografi, MRI, är en användbar teknik inom flera områden, i denna uppsats ligger fokus på användning inom medicin. Fysiken som ligger bakom MRI presenteras, som t ex uppdelning av energinivåer och kärnmagnetiskresonans. Uppbyggnad och tekniken som ligger bakom MRI har även studeras. Sedan har det gjorts jämförelse mellan MRI, röntgen och datortomografi.   Utöver litteraturstudierna har simulering gjorts. I denna del har en modell för hjärnan byggts i programmet Comsol Multiphysics. I denna modell har studier av energiöverföringen, magnetiska fältet och elektriska fältet gjorts. Modellen har lösts både som stationärt problem och som tidsberoende. För energiöverföringen som redovisas som tidsmedelvärdet ser resultaten liknade ut för det stationära fallet som för de tidsberoende fallen, dock om man inte ser på tidsmedelvärdet utan energiöverföring under hela tiden så kan man dra slutsatsen att det stationära fallet är det mest kritiska. / <!-- /* Font Definitions */ @font-face {font-family:SimSun; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-alt:宋体; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} @font-face {font-family:"\@SimSun"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:134; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:1 135135232 16 0 262144 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:SimSun; mso-fareast-language:ZH-CN;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 70.85pt 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} -->Magnetic resonance imaging, MRI, is a useful technical method in many different areas; in this report lies focus on uses in medicine. MRI has been studied from a physical meaning, such as nuclear magnetic resonance, NMR, and spin splitting. A technical perspective of MRI has been studied, such as how MRI is built-up and technical details. MRI has been compared with Computed Tomography, CT, and X-rays.   In addition to the theoretical studies, have simulations using the programme Comsol Multiphysics been done. One model has been built up to simulate MRI influences on the brain. The energy transfer, magnetic field and electric field have been studied. The model has been solved both as stationary and as time dependent problem. In the solution can a small difference be noticed which depend on that the results show the time average. If one studies the real solution, not the time average, can one conclusion rather quickly been drawn that the stationary solution have the highest transferred energy.

The extensive agricultural usage of organohalogenated pesticides has raised many concerns about their potential hazards especially in the soil environment. Environmental metabolomics is an emerging field that investigates the changes in the metabolic profile of native organisms in their environment due to the presence of an environmental stressor. Research presented here explores the potential of Nuclear Magnetic Resonance (NMR)-based metabolomics to examine the sub-lethal exposure of the earthworm, Eisenia fetida to sub-lethal concentrations of organohalogenated pesticides. Various one-dimensional (1-D) and two dimensional (2-D) NMR techniques were compared in a contact filter paper test earthworm metabolomic study using endosulfan, a prevalent pesticide in the environment. The results determined that both the 1H Presaturation Utilizing Gradients and Echos (PURGE) and the 1H-13C Heteronuclear Single Quantum Coherence (HSQC) NMR techniques were most effective in discriminating and identifying significant metabolites in earthworms due to contaminant exposure. These two NMR techniques were further explored in another metabolomic study using various sub-lethal concentrations of endosulfan and an organofluorine pesticide, trifluralin to E. fetida. Principal component analysis (PCA) tests showed increasing separation between the exposed and unexposed earthworms as the concentrations for both contaminants increased. A neurotoxic mode of action (MOA) for endosulfan and a non-polar narcotic MOA for trifluralin were delineated as many significant metabolites, arising from exposure, were identified. The earthworm tissue extract is commonly used as the biological medium for metabolomic studies. However, many overlapping resonances are apparent in an earthworm tissue extract NMR spectrum due to the abundance of metabolites present. To mitigate this spectral overlap, the earthworm’s coelomic fluid (CF) was tested as a complementary biological medium to the tissue extract in an endosulfan exposure metabolomic study to identify additional metabolites of stress. Compared to tests on the tissue extract, a plethora of different metabolites were identified in the earthworm CF using 1-D PURGE and 2-D HSQC NMR techniques. In addition to the neurotoxic MOA identified previously, an apoptotic MOA was also postulated due to endosulfan exposure. This thesis also explored the application of 1-D and 2-D NMR techniques in a soil metabolomic study to understand the exposure of E. fetida to sub-lethal concentrations of endosulfan and its main degradation product, endosulfan sulfate. The earthworm’s CF and tissue extract were both analyzed to maximize the significant metabolites identified due to contaminant exposure. The PCA results identified similar toxicity for both organochlorine contaminants as the same separation, between exposed to the unexposed earthworms, were detected at various concentrations. Both neurotoxic and apopotic MOAs were observed as identical fluctuations of significant metabolites were found. This research demonstrates the potential of NMR-based metabolomics as a powerful environmental monitoring tool to understand sub-lethal organohalogenated pesticide exposure in soil using earthworms as living probes.

This thesis is about excitation, detection, properties and applications of multiple quantum coherences applied to different dipolar coupled spin systems. Major focus of the work is on spectral simplification, measurement of residual dipolar couplings and discrimination of enantiomers in chiral aligning media. The first chapter gives a brief account on the fundamentals of nuclear magnetic resonance spectroscopy and multiple quantum coherences. This includes a description of product operator and polarization operator formalisms of pulses and evolution of magnetization. Subsequently a detailed account of two dimensional multiple quantum – single quantum (MQ-SQ) correlation experiments is given. Demonstration of the homonuclear MQ-SQ pulse sequence on a weakly coupled spin system and analysis of the spectrum obtained are also discussed. Homo-nuclear multiple quantum studies carried out to obtain relative the signs of the couplings have been reported in the initial part of the second chapter. The technique has been applied on doubly labeled acetonitrile (13CH313C15N) aligned in a liquid crystalline medium. Special situations like ambiguity in the determination of relative signs of the couplings from the appearance of two dimensional MQ-SQ spectra and the explanation for the same are also discussed. Homo-nuclear MQ experiments on indistinguishable spins, like protons in a methyl group of 13CH313C15N oriented in liquid crystal, and distinguishable spins, like the two carbons in the same molecule, have been carried out. Different directions of approach in which these results need to be analyzed have been discussed. Subsequent part of the chapter is about the correlation of connected MQ-SQ coherences. These experiments are significant in reducing the cross-peaks further from the MQ-SQ spectra. This concept is extended for the discrimination of optical enantiomers dissolved in chiral aligning medium made of poly-Γ-benzyl-L-glutamate (PBLG) and CDCl3. In molecules of Chemical and biological interest one encounters several nuclei such as, 1H, 13C, 15N and 19F. It will be of general interest to determine magnitudes and relative signs of the couplings among these coupled nuclei by NMR experiments. Utilization of hetero-nuclear MQ Experiments in solving such problems is discussed in the third Chapter. Hetero-nuclear MQ experiments were carried out on dipolar coupled 13CH313C15N, with the aim of obtaining the values and signs of various hetero-nuclear couplings in the molecule. The splitting of transitions in the spectra of oriented molecules is always influenced by the sum of dipolar and scalar couplings. Hence precise determination of dipolar couplings requires the knowledge of scalar couplings. To determine the J couplings, experiments were carried out on the same molecule in isotropic medium. When many coupled nuclei are involved one has to carry out several experiments to derive all the spectral parameters. In circumventing this problem heteronuclear multiple quantum experiments involving more than two nuclei as active spins are advantageous. This reduces the number of experiments and thereby reducing the total experimental time. Second part of this chapter demonstrates how a triple resonance triple quantum experiment can provide majority of the couplings from a given coupled system. The feasibility of the experiment is demonstrated even for molecules containing natural abundant isotopes. Application of multiple quantum j-resolved technique for chiral discrimination and obtaining complete one dimensional spectrum of each enantiomer from their racemic mixture is discussed in the fourth chapter. The two dimensional experiment consists of a selective double quantum excitation period followed by selective refocusing during indirect time domain, isotropic mixing and nonselective detection of SQ transitions. Hence this pulse sequence is named as DQSERF-COSY (Double Quantum Selective Refocused Correlation Spectroscopy). The experiment exploits the existence of different intra-methyl couplings between the enantiomers dissolved in chiral liquid crystal medium to separate the one dimensional spectra of each enantiomer in different cross sections. This is possible due to the fact that all the nuclei in any one of the enantiomers are coupled among themselves and there is no inter molecular interaction between the two enantiomers. Also one can extract all the couplings between protons in each enantiomer, which can subsequently be utilized for determination of the residual dipolar couplings, structure and orientation parameters.

<p>This dissertation focuses on a very special topic in the field of Nuclear Magnetic Resonance (NMR) in solution: Intermolecular Multiple Quantum Coherences, or iMQCs, which can only be created by intermolecular dipolar couplings. Since the very beginnings of NMR, it has been known that dipolar couplings dominate the solid-state linewidth for spin-1/2 nuclei, but the effects are still not fully understood. The angular dependency (1-3cos2&#952;ij) and distant dependency (rij-3) of dipolar coupling led to an oversimplified conclusion that it can be ignored in an isotropic liquid. Thus, it was surprising when COSY Revamped by Asymmetric Z-gradient Echo Detection (CRAZED) was first introduced in the early `90s and showed strong iMQC signals. Since then, CRAZED has inspired a wide range of applications for iMQCs and led to two different but equivalent mathematical frameworks to describes these effects, which we call the conventional DDF theory.</p><p>However, several disagreements between the conventional DDF theory and experiments have grasped our attention recently. This dissertation will: first, demonstrate how conventional picture fails by two examples, Multi-axis CRAZED (MAXCRAZED) and Gradient-embedded COSY Experiment (GRACE); second, provide a corrected DDF theory; and, third, discuss what impact this correction will bring.</p><p>Intermolecular double quantum coherences (iDQCs) are very sensitive to the local anisotropy (10&#956;m - 1mm) and can be used to create positive contrast highlighting superparamagnetic iron oxide nanoparticles (SPIONs). This dissertation will show the design and optimization of iDQC anisotropy by a series of phantom experiments. A set of numerical simulations will then be provided for a sub-voxel level explanation. We will also demonstrate how the newly corrected DDF theory can be quickly adapted to improve the iDQC anisotropy.</p><p>Finally, as a side product of this research, the mechanism of diacetyl hydration/dehydration as solved by NMR will be provided.</p> / Dissertation

Implanted tissue engineered substitutes constitute dynamic systems, with remodeling mediated by both the implanted cells and the host. Thus, there exists a significant need for methods to monitor the function and morphology of tissue engineered constructs. Noninvasive monitoring using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and imaging can prove to be the solution to this problem. Spectroscopy allows for assessment of cellular function through the monitoring of inherent metabolic markers, such as total-choline, while high resolution imaging enables the evaluation of construct morphology and interfacial remodeling. We applied these 1H NMR methods to monitor betaTC3 mouse insulinoma cells within hydrogel-based materials as a model pancreatic tissue substitute. In vitro research established a strong correlation between total-choline, measured by 1H NMR spectroscopy, and viable betaTC3 cell number, measured by MTT. Extending these methods to in vivo monitoring, however, was met with additional challenges. First, the implanted cells needed to be contained within a planar construct above a threshold density to allow for adequate quantification of the total-choline peak. Secondly, cell-free buffer zones between the implanted cells and the host tissue needed to be incorporated to prevent host tissue signal contamination. Finally, quantitative techniques needed to be developed to accurately account for contaminating signal from diffusing molecules. To overcome these challenges, a disk-shaped agarose construct, initially containing a minimum of 4 million betaTC3 cells and coated with an outer layer of pure agarose, was fabricated. Mathematical simulations aided the implant design by characterizing diffusive transport of nutrients and metabolites into and out of the construct. In vivo 1H NMR studies of these constructs implanted in mice established a strong correlation between total-choline, measured noninvasively using 1H NMR spectroscopy, and viable cell number, measured invasively using MTT. This study establishes total-choline as a reliable marker for noninvasively quantifying dynamic changes in viable betaTC3 cell number in vivo. 1H NMR imaging was used to monitor the implants structural integrity over time, while also assessing the hosts fibrotic response. We expect these studies to establish quantitative criteria for the capabilities and limitations of NMR methodologies for monitoring encapsulated insulinomas, as well as other tissue implants.

No description available.

The present work, briefly described below, consists of implementation of several quantum algorithms in an NMR Quantum Information Processor. Game theory gives us mathematical tools to analyze situations of conflict between two or more players who take decisions that influence their welfare. Classical game theory has been applied to various fields such as market strategy, communication theory, biological processes, foreign policies. It is interesting to study the behaviour of the games when the players share certain quantum correlations such as entanglement. Various games have been studied under the quantum regime with the hope of obtaining some insight into designing new quantum algorithms. Chapter 2 presents the NMR implementation of three such algorithms. Experimental NMR implementation given in this chapter are: (i) Three qubit ‘Dilemma’ game with corrupt sources’. The Dilemma game deals with the situation where three players have to choose between going/not going to a bar with a seating capacity of two. It is seen that in the players have a higher payoff if they share quantum correlations. However, the pay-off falls rapidly with increasing corruption in the source qubits. Here we report the experimental NMR implementation of the quantum version of the Dilemma game with and without corruption in the source qubits. (ii) Two qubit ‘Ulam’s game’. This is a two player game where one player has to find out the binary number thought by the other player. This problem can be solved with one query if quantum resources are used. This game has been implemented in a two qubit system in an NMR quantum information processor. (iii) Two qubit ‘Battle of Sexes’ game. This game deal with a situation where two players have conflicting choices but a deep desire to be together. This leads to a dilemma in the classical case. Quantum mechanically this dilemma is resolved and a unique solution emerges. The NMR implementation of the quantum version of this game is also given in this chapter. Quantum adiabatic algorithm is a method of solving computational problems by evolving the ground state of a slowly varying Hamiltonian. The technique uses evolution of the ground state of a slowly varying Hamiltonian to reach the required output state. In some cases, such as the adiabatic versions of Grover’s search algorithm and Deutsch-Jozsa algorithm, applying the global adiabatic evolution yields a complexity similar to their classical algorithms. However, if one uses local adiabatic evolutions, their complexity is of the order √N (where N=2n) [37, 38]. In Chapter 3, the NMR implementation of (i) the Deutsch-Jozsa and the (ii) Grover’s search algorithm using local adiabatic evolution has been presented. In adiabatic algorithm, the system is first prepared in the equal superposition of all the possible states which is the ground state of the beginning Hamiltonian. The solution is encoded in the ground state of the final Hamiltonian. The system is evolved under a linear combination of the beginning and the final Hamiltonian. During each step of the evolution the interpolating Hamiltonian slowly changes from the beginning to the final Hamiltonian, thus evolving the ground state of the beginning Hamiltonian towards the ground state of the final Hamiltonian. At the end of the evolution the system is in the ground state of the final Hamiltonian which is the solution. The final Hamiltonian, for each of the two cases of adiabatic algorithm described in this chapter, are constructed depending on the problem definition. Adiabatic algorithms have been proved to be equivalent to standard quantum algorithms with respect to complexity [39]. NMR implementation of adiabatic algorithms in homonuclear spin systems face problems due to decoherence and complicated pulse sequences. The decoherence destroys the answer as it causes the final state to evolve to a mixed state and in homonuclear systems there is a substantial evolution under the internal Hamiltonian during the application of the soft pulses which prevents the initial state to converge to the solution state. The resolution of these issues are necessary before one can proceed for the implementation of an adiabatic algorithm in a large system. Chapter 4 demonstrates that by using ‘strongly modulated pulses’ for creation of interpolating Hamiltonian, one can circumvent both the problems and thus successfully implement the adiabatic SAT algorithm in a homonuclear three qubit system. The ‘strongly modulated pulses’ (SMP) are computer optimized pulses in which the evolution under the internal Hamiltonian of the system and RF inhomogeneities associated with the probe is incorporated while generating the SMPs. This results in precise implementation of unitary operators by these pulses. This work also demonstrates that the strongly modulated pulses tremendously reduce the time taken for the implementation of the algorithm, can overcome problems associated with decoherence and will be the modality in future implementation of quantum information processing by NMR. Quantum search algorithm, involving a large number of qubits, is highly sensitive to errors in the physical implementation of the unitary operators. This can put an upper limit to the size of the data base that can be practically searched. The lack of robustness of the quantum search algorithm for a large number of qubits, arises from the fact that stringent ‘phase-matching’ conditions are imposed on the algorithm. To overcome this problem, a modified operator for the search algorithm has been suggested by Tulsi [40]. He has theoretically shown that even when there are errors in implementation of the unitary operators, the search algorithm with his modified operator converges to the target state while the original Grover’s algorithm fails. Chapter 5, presents the experimental NMR implementation of the modified search algorithm with errors and its comparison with the original Grover’s search algorithm. We experimentally validate the theoretical predictions made by Tulsi that the introduction of compensatory Walsh-Hadamard and phase-flip operations refocuses the errors. Experimental Quantum Information Processing is in a nascent stage and it would be too early to predict its future. The excitement on this topic is still very prevalent and many options are being explored to enhance the hardware and software know-how. This thesis endeavors in this direction and probes the experimental feasibility of the quantum algorithms in an NMR quantum information processor.

Branched chain amino acids are classified as essential amino acids since their biosynthetic routes or pathways are restricted only to micro-organisms, fungi and plants. Given their unique distribution, the enzymes of the branched chain amino acid biosynthetic pathway are ideal targets for the development of herbicides, anti-bacterials and potentially antifungal agents. Acetohydroxy acid synthase (AHAS) catalyses the firs step in the biosynthesis of branched chain amino acids. AHAS activity had been first identified in extracts of E. coli as early as in 1958 by Brown and Umbarger . Ever since its discovery, AHAS have been found to exist in all eubacteria, archaebacteria, algae, yeast and plants. The enzymatic properties of prokaryotic and eukaryotic AHASs have been thoroughly investigated. A single isoform of the enzyme is known to exist in all organisms except in enterobacteria which have three isoforms of the enzyme. Activity of the three isoforms of E. coli AHAS (I, II and III) have been studied using various biochemical and biophysical methods. AHAS enzyme expressed in bacteria and yeast are heterotetrameric composed two large catalytic and two small regulatory subunits. While much has been learnt from the structure of the catalytic subunits (yeast and Arabidopsis thaliana) and the regulatory subunits (regulatory subunit of E. coli AHAS III) in isolation, the structural properties of the holoenzyme remain unexplored. AHAS is unique from the point of view that it exhibits a striking domain organization in the catalytic subunit and also in the regulatory subunits. Thus understanding the nature of protein – protein interactions both as domain – domain interactions within the subunit as well as protein – protein interactions across subunits is crucial to understanding the structural basis for the activity and regulation of this important enzyme. Of these, understanding the structural basis for the interaction between the regulatory and the catalytic subunits within the holoenzyme is paramount. The poor solubility and the intrinsic instability of the proteins have hampered the efforts to structurally characterize any of the AHAS holoenzymes. An active AHAS I construct has been created by Vyazmensky et. al., where the catalytic and the regulatory subunit have been expressed together as a single chain separated by a flexible linker. While this single chain construct is catalytically active, there have been no reports of successful crystallization of this single chain AHAS I enzyme. The crystallographically determined structure of the catalytic subunit of yeast and A. thaliana AHAS has shown that the protein is composed of three independently folded domains, α, β and γ. More importantly the polypeptide sequence of the catalytic subunits of AHAS across all species is largely conserved. This indicates that the overall tertiary folds of the catalytic subunit would be alike. The unique domain architecture of the AHAS catalytic subunit and the relatively small size of the regulatory subunit forms the basis for implementation of a novel strategy, in which structural interactions between the domains (catalytic site as well as the non catalytic site interactions) as well as structural interactions between the domains of the catalytic and the regulatory subunit of E. coli AHAS I can be explored in an incremental manner. Initiation of structural characterization of the individual domains of the catalytic subunit of E. coli AHAS I and understanding the structural basis of the interaction between the domains of the catalytic and the regulatory subunits of the protein, using solution NMR methods, forms the theme for this study. The domains of the catalytic subunit (ilvB) of E.coli AHAS I were identified based on the similarity in the sequence of this subunit with the yeast protein and the structural information of the yeast protein. The individual domains of the ilvB protein (ilvBα, ilvBβ and ilvBγ) and ilvN, the regulatory subunit of AHAS I, were cloned, expressed and purified for structural studies. The problem of poor expression and solubility profiles of the AHAS proteins was circumvented with the help of a novel cytb5 fusion system developed in our laboratory during the course of this study. The high expression levels of the fusion protein in minimal medium enabled the preparation of isotopically (15N, 13C/15N, 2H/13C/15N) enriched samples of the proteins in a cost effective manner. The cytb5 fusion system has provided very uniform and reliable expression of these proteins without accumulation of any protein in the insoluble fraction. From the structure of the catalytic subunit of yeast AHAS it is known that the α and γ domains of the protein interact to form the active site. The two domains provide group specific interaction sites for anchoring the co-factor TPP in an appropriate conformation for catalysis. The β domain on the other hand does not directly participate in the ormation of the active site but anchors the co-factor FAD which in turn plays a structural role in enzyme catalysis. In the present study we employed biochemical and biophysical methods to establish the structural integrity of the individually expressed domains of the catalytic subunit (ilvB) and the regulatory subunit of AHAS I. Reactions catalyzed by enzymes formed by assembling different domain and subunits indicate that the proteins when reconstituted in vitro form a catalytically competent complex. Formation of S-acetolactate, the product of the reaction catalyzed by the AHAS I holoenzyme, has been confirmed using colorimetric as well as spectroscopic methods such as CD and NMR. Multinuclear, multidimensional NMR methods have been utilized to obtain sequence specific assignments of apo - ilvBβ (non FAD bound form). Preparation of an NMR amenable sample of ilvBβ proved to be the rate limiting step due to the predisposition of the protein to undergo aggregation at concentrations required for solution NMR studies. However, careful screening of large number of buffer conditions enabled us to establish an optimum sample condition where the protein was soluble, stable and free of aggregation and hence suitable for NMR studies. Uniformly enriched 15N, 13C/15N, and 2H/13C/15N samples of ilvBβ were prepared to obtain sequence specific assignments and secondary structural information. From the secondary chemical shifts of backbone 13Cα atoms and short and medium range NOEs the secondary structure of the non FAD bound (apo) form of ilvBβ has been determined. Using chemical shift mapping methods, the residues of the ilvBβ domain that are involved in FAD binding have been identified. The distribution of the secondary structural elements and the residues that are involved in binding the co-factor FAD were found to be conserved for the E. coli and yeast proteins. This suggests that the tertiary Fold of the FAD binding β domain of the catalytic subunit of E. coli AHAS is identical to that in the yeast protein. The interaction between the individual domains of ilvB and ilvN (the regulatory subunit) has been investigated using spectroscopic methods. Changes in CD spectra indicate that ilvN interacts with ilvBα and ilvBβ domains of the catalytic subunit and not with the ilvBγ domain. NMR chemical shift mapping methods has shown that ilvN binds close to the FAD binding site in ilvBβ and proximal to the intra-subunit ilvBα/ilvBβ domain interface. The implication of this interaction and the role of the regulatory subunit on the activity of the holoenzyme are discussed.