This article reviews the properties and applications of a series of liquid crystal solvents for NMR spectroscopy of, particularly, biological compounds with emphasis placed on n-alkyl-poly(ethylene glycol)/n-alkyl alcohol, the most versatile liquid crystal system hitherto discovered. The applications of this solvent to mixtures, polymers and proteins are also mentioned.

NMR studies have long been used as a tool to derive structural and dynamic information. Such information has a wide range of applications, and notably is used in the study of structure-activity relationships. The aims of this work were to use NMR spectroscopy to derive structures of the molecules inhibiting the activation of the alternative pathway of the complement portion of the innate immune system (namely, the N-terminus of factor H (FH) and two small peptides, Compstatin 10 and Compstatin 20) and to consider the interdomain dynamics of proteins consisting of three modules theoretically (in silico) and experimentally (for the three N-terminal domains of FH). We focused on the three N-terminal complement control protein (CCP) domains of the important complement regulator, human factor H (i.e. FH1-3). Its three-dimensional solution structure was derived based on nuclear Overhauser effects and residual dipolar couplings (RDCs). Each of the three CCP modules in this structure was similar to the corresponding CCP in the previously derived C3b-bound structure of FH1-4, but the relative orientations of the domains were different. These orientations were additionally different from the interdomain orientations in other molecules that interact with C3b, such as DAF2-4 and CR1-15-17. The measured RDC datasets, collected under three different conditions in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids), were used to estimate interdomain motions in FH1-3. A method in which the data was fitted to a structural ensemble was used to analyze such interdomain flexibility. More than 80% of the conformers of this predominantly extended three-domain molecule exhibit flexions of < 40°. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3) could facilitate recognition of C3b via initial anchoring, as well as eventual reorganization of modules into the conformation captured in the previously solved crystal structure of a C3b complex with FH1-4. The NMR study of the Compstatin analogues revealed unique structural features that had not before been observed in this group of peptides. These features included two b-turns per peptide, neither of which was located in the ‘canonical’ regions in which b-turns were observed in previous molecular dynamics and NMR studies. The structures of Compstatin 10 and Compstatin 20 derived here were consistent with the isothermal calorimetry (ITC) and surface plasmon resonance (SPR) data recorded previously. In the in silico study of interdomain motion of three-domain proteins carried out here, the domains were represented as vectors attached to one another in a linear fashion. They were allowed to undergo Brownian motion biased by the potentials between the sequential vectors. The resulting trajectories were analyzed using model-free and extended model-free formalism. The degree of coupling of the interdomain motion with overall motion was determined, along with a representation of the overall motion. The similarity between the trajectories of the vectors transformed to this overall motion frame and the results obtained from the model-free analysis was determined.

Carbohydrates play important roles in biological processes. Their function is closely related to their conformation. In this thesis, conformational studies of carbohydrates by NMR spectroscopy and molecular dynamics computer simulations are described. The first two papers discuss the anomalous solubility of β-cyclodextrin compared to other cyclodextrins. Time correlation functions revealed flexibility in all cyclodextrins. Molecular dynamics computer simulations showed that the glycosidic linkages were rather rigid and the flexibility was suggested to be macrocyclic. From spatial distribution functions β-cyclodextrin was found to have greater ability to order the surrounding water than the other cyclodextrins. Paper III deals with some of the difficulties of conformational studies. In Paper IV, a new method, Additative Potential Maximum Entropy, APME, is applied to a disaccharide. Conformational distribution functions are derived from NOEs, J-couplings and residual dipolar couplings and calculated from computer simulations. All distribution functions were found to be in good agreement. In papers V and VI oligosaccharides from human milk are studied. Residual dipolar coupling, J-couplings and cross relaxation rates were measured by NMR spectroscopy and molecular dynamics computer simulations were carried out. Both oligosaccharides showed high flexibility for the β-D-GlcpNAc-(1→3)-β-D-Galp linkage.

<p>In this work, I develop computational and visual tools for analyzing and manipulating the backbone of macromolecules, and I demonstrate that these tools support building better structures than currently done. These visualization and analysis tools belong to an "Intelligence Amplification" (IA) tradition (rather than complete Artificial Intelligence (AI) automation), empowering users to improve structures.</p><p>Proteins and nucleic acids are among the most important molecules in biology, mediating the majority of biochemical processes that comprise a living organism. Therefore, these macromolecules are important targets, both for basic research to improve understanding of how life works, and for medical research as possible drug targets. </p><p>The function of these macromolecules is largely determined by their 3D structure. Although these macromolecules are chemically fairly simple, made up of linear sequences of a few possible subunits, they physically fold into complex, compact structures. Overall, structural biology aims to determine the general relationship between sequence and structure of these macromolecules, through determination of the positions of the atoms within individual macromolecules. </p><p>Because it is currently impossible to directly see the position of atoms in a molecule, all structural determination techniques, including X-ray crystallography, NMR, and homology modeling, result in an interpreted <italic>model</italic> of a structure. Nearly all of these models contain mistakes, in which atoms are fit in incorrect or impossible positions. These mistakes, especially at a functionally-important location in a structure, can mislead both basic and medical research, making it critical for structural biologists to build the highest quality models possible. </p><p>This document details how my dissertation work enables the building of better macromolecular structure models. This work follows an iterative development cycle, where visual analysis of models spurs development of better tools, which in turn improves the analysis. First, I describe how my analysis of protein loops from X-ray crystal structures reveals that the traditional definition of loop endpoints is too restrictive. Second, I create a protein backbone analysis and modeling tool, using a new peptide-centric division system. I show how this tool makes it easier to study protein loops, and also how it improves an algorithm for calculating core protein models from NMR residual dipolar coupling (RDC) data. Third, I describe how 3D visualization of RDCs in their structural context improves understanding of RDCs and validates NMR models in a novel way. Fourth, I describe how local quality analysis can diagnose problems in homology models. Fifth, I demonstrate that local quality analysis can be successfully used in conjunction with model rebuilding software to correct errors in low resolution structures. The various tools and software packages I created during the course of my work are freely available and have already made a positive impact on structures being generated by the community.</p><p>Archive versions of several of these software packages (JiffiLoop, RDCvis, and KiNG) should be included with this document; current versions can be downloaded from http://kinemage.biochem.duke.edu.</p> / Dissertation

The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of MBD2 and MBD3. This study provides information regarding previously unidentified MBD3 binding properties and potential biological function. In chapter three, we show that sponges demonstrate a MBD2-mediated capacity for binding methylated DNA sites, recruit NuRD components in vitro, and knockdown of MBD2 in the freshwater desmosponge, Ephydatia muelleri, promotes an abnormal growth phenotype.

La spectroscopie RMN alliée à l’utilisation de solvants cristal-liquide fortement et faiblement orientants est une stratégie efficace pour élucider les structures et distributions conformationnelles de petites molécules organiques rigides et flexibles en solution, et déterminer les ordres orientationnel et positionnel des solutés comme des solvants orientés. Dans une première partie, afin d’explorer les différentes contributions aux couplages dipolaires d’un soluté donné, la très faible amplitude de l’ordre orientationnel d’une molécule quasi-sphérique, le tetramethylallène, dissoute dans un nématique thermotrope est exploitée. Dans cette situation limite, le caractère prédominant des mécanismes de réorientation et de vibration moléculaire est mis en évidence, et estimé. Dans une seconde partie, les données RMN obtenues à partir de solutés de petites tailles dissous dans des solvants smectiques sont combinées aux résultats de calculs reposant sur des concepts de thermodynamique statistique et de la théorie de la fonctionnelle de densité. L’efficacité de cette méthode dans la détermination des paramètres d’ordres positionnel du solvant et orientationnel des molécules-sondes est démontrée aussi bien dans le cas de phases conventionnelles smectiques A que celui plus délicat de smectiques interdigitées Ad. La stratégie d’analyse proposée est ensuite étendue à l’investigation des structures tridimensionnelles et équilibres conformationnels de molécules flexibles bioactives ou biomimétiques. Dans une perspective méthodologique, à l’aide d’études expérimentale et théorique portant sur le biphényle, molécule symétrique constituée d’un unique rotor, il est tout d’abord démontré l’intérêt des méthodes de simulations par dynamique moléculaire pour évaluer l’ensemble des couplages dipolaires d’un soluté donné dans une phase thermotrope, ultérieurement utilisés comme paramètres initiaux dans une analyse spectrale itérative, et in fine déterminées précisément. L’analyse spectrale chronophage et dont l’aboutissement est incertain si les paramètres initiaux sont difficiles à estimer, en est ainsi facilitée. Puis, les distributions conformationnelles d’anti-inflammatoires non stéroïdiens de dérivés salicylés et profènes, fluorés ou non, constitués d’un ou deux rotors indépendants sont présentées. Via l’utilisation inédite du modèle AP-DPD dans les solvants nématiques (chiraux) lyotropes faiblement orientants, et à partir des couplages dipolaires homo- et hétéronucléaires notamment obtenus grâce à l’expérience RMN GET-SERF, créée à propos pour permettre l’extraction simple et rapide des couplages 1H-19F, les surfaces d’énergie potentielle de ces biomolécules sont décrites de façon satisfaisante. Enfin, les équilibres conformationnels de deux stilbénoïdes constitués de deux rotors coopératifs sont déterminés dans deux solvants cristal-liquide, l’un fortement, l’autre faiblement orientant. Ces études comparatives permettent de discuter la fiabilité, la précision et l’accessibilité des observables RMN extraites dans les phases, et d’établir la complémentarité des analyses RMN réalisées dans ces solvants. / NMR spectroscopy in weakly and highly orienting media is used as a route for dealing with orientational, positional, structural and conformational problems of a variety of small rigid and flexible organic molecules in solution. First, the very weak orientational order of a quasi-spherical molecule dissolved in a nematic phase is exploited for exploring the role of the different contributions to the observed dipolar coupling. In such a limit condition, a predominant effect of the non-rigid reorientation-vibration coupling term emerges. Then, NMR data obtained from small rigid probes dissolved in smectic solvents are combined with a statistical thermodynamic density functional theory, in order to measure the positional order parameters of both solutes and solvent. The methodology gives good results when applied to a conventional smectic A liquid crystal and to the more delicate case of an interdigitated smectic Ad phase. The strategy is subsequently extended to the investigation of structure, order and conformational equilibrium of flexible bioactive or biomimetic molecules dissolved in various partially ordered NMR solvents. A first experimental and theoretical study is presented on the symmetric single-rotor molecule of biphenyl dissolved in a thermotropic liquid crystal. This test-case indicates molecular dynamics simulations are a promising tool for estimating a set of dipolar couplings of a solute in a thermotropic solvent, to be used as starting set of parameters in a standard operator-mediated NMR spectral analysis. Then, we report the conformational study of some single- and two-rotor nonsteroidal anti-inflammatory drugs, belonging to the families of salicylates and profens, dissolved in weakly orienting chiral nematic PBLG phases. A new pulse sequence, the Gradient Encoded heTeronuclear 1H-19F SElective ReFocusing NMR experiment (GET-SERF), is proposed here for the trivial edition of all 1H-19F couplings in one single NMR experiment, for a given fluorine atom. Starting from homo- and heteronuclear dipolar couplings, difficult to extract in thermotropic solvents because of a too complex spectral analysis, the torsional distributions of such molecules can be satisfactory described by the Additive Potential model combined with the Direct Probability Description of the torsional distribution in terms of Gaussian functions (AP-DPD approach). Finally, the conformational and orientational study of two stilbenoids displaying cooperative torsions is discussed in both a highly and weakly ordering liquid crystal phase. This comparative study allows to draw some conclusions on reliability, accuracy and accessibility of desired data in the two phases. Overall, this work proves NMR in liquid crystals is a flexible and meaningful tool for studying order, structure and conformation and it can greatly benefit from the availability of several aligning media inducing a different degree of order.

The thesis aims at methodological developments in Nuclear Magnetic Resonance (NMR) and study of oriented samples like liquid crystals and single crystals and powder samples. Though methodological development in solid state NMR (ss-NMR) has gone far ahead, this work attempts to contribute some novel methods in this direction. The work presented here falls into two categories (i) methodological developments for obtaining information on anisotropic interactions and (ii) experiments which utilize the existing methodologies to study systems of interest under static condition and also under sample spinning at the Magic Angle. In the solid state, dipolar couplings play a crucial role. On the one hand these couplings could be used to transfer polarization from an abundant nucleus to a rare nucleus and increase the sensitivity of the rare nucleus. On the other hand, the measurement of dipolar couplings itself is crucial for extracting structural and dynamic information. A third aspect is that dipolar couplings could be used to obtain correlation, say between two different nuclear species or between the same kind of nuclei as in an exchange experiment. A major part of this thesis deals with all three aspects mentioned above. The thesis presents a new heteronuclear polarization transfer scheme which is devoid of some of the short comings of the existing and well-known polarization transfer schemes. This pulse sequence has been found to be useful in different contexts involving both spin ½ and spin 1 nucleus. The use of dipolar couplings for obtaining correlation in both static oriented systems and in powder samples has been illustrated. In the case of the powder sample, the study has been useful in obtaining useful orientation information. Finally, chemical shifts are known to be indicators of finer structural features of molecules in solution and solid state. 13 C MAS NMR studies have been exploited in understanding these structural features of short peptides containing prolines in the solid state and for comparing with their structures in solution. Chapter 1 covers the theoretical aspects required for the experimental work described in the thesis. A brief description of NMR has been followed by the explicit description of various interaction Hamiltonian’s in ss-NMR. Subsequently the experimental and the theoretical tools needed for ss-NMR study like Magic Angle Spinning (MAS), Cross-Polarization, Homo/Heteronuclear decoupling schemes have also been discussed. Chapter 2, describes a new heteronuclear polarization transfer scheme for oriented samples – named DAPT (Dipolar Assisted Polarization Transfer) and its application to different systems. DAPT uses a homonuclear decoupling sequence such as BLEW-12 for effecting heteronuclear polarization transfer. The chapter has been divided into five related parts. Section 2(A) starts with an introduction to the existing heteronuclear polarization transfer schemes. Subsequently the theoretical background of the new sequence is presented. Experimental implementation of the sequence in an oriented system, liquid crystal is presented and is compared with the well-known polarization transfer scheme, Hartmann-Hahn Cross Polarization (HH-CP). In 2(B) the implementation of the sequence as a local field spectroscopy for measuring heteronuclear dipolar couplings is presented. After initial discussion about local field spectroscopy and its relevance in ss-NMR, the improvements made in the earlier mentioned sequence along with its 2D implementation in a liquid crystal sample are described. A comparative study is also presented using DAPT with various other homonuclear decoupling sequences. Chapter 2(C) deals with the extension of DAPT to spin-1 systems. The difficulties in setting up the HH-CP in spin-1 systems are highlighted. Experimental demonstrations on a test sample of oriented CD3 I and also on a deuterated liquid crystal is described. The sequence has been incorporated as part of a 2D correlation experiment, where the F1 dimension provides the quadrupolar couplings of deuterium and the F2 the chemical shifts of the attached carbons. The comparison of the sequence with HH-CP, its merits and demerits are discussed and the potential applications are highlighted. Chapter 2(D) deals with the relatively less studied transition in 14N nucleus, known as the Overtone-Transition (OT). An introduction to OT and its relevance is provided in the beginning followed by the extension of DAPT in exciting and detecting OT. The experiments have been done on a single crystal of a model peptide, N-Acetyl-DL-Valine and are compared with the conventional method. Amide proton chemical shifts are also measured using DAPT in an indirect way. The advantages and the future application in studying OT are also discussed. Chapter 2(E) discusses the extension of DAPT to the single crystal of NAV and in identifying the molecules in the unit cell. The SLF spectrum of NAV is complicated due to the presence of two magnetically in-equivalent molecules in the unit cell and with pairs of splitting for each C - 1H and C - 1H pairs. The dipolar couplings are extracted from the experiment and with the aid of a MATLAB program and by incorporating the crystal coordinates, identification of C-1H and C-1H pairs belonging to a particular molecule have been carried out. Chapter 3 describes a novel and useful modification of the well-known Separated Local Field (SLF) sequence in solid state known as PISEMA (Polarization Spin Exchange at the Magic Angle). PISEMA is a popular technique for measuring heteronuclear dipolar couplings in oriented in oriented biological membranes and in liquid crystals. While it has several advantages such as a large dipolar scaling factor, narrow line-widths in the dipolar dimension and ease of setting up etc it suffers from a major problem. The technique is highly sensitive to the proton off-sets which affect the measured dipolar couplings. In the present chapter the origin of this problem has been analyzed in detail and a solution has been proposed. The modification to the experiment has been implemented on a liquid crystal and the off-set independence of the new sequence has been demonstrated. Further studies on a more rigid system such as a solid single crystal has been used to verify the effect of the modification on homonuclear decoupling efficiency and the consequent effects on the line widths in addition to off-set independence. The advantages of the proposed method over the existing one in terms of line-width and robustness in measuring heteronuclear dipolar couplings are demonstrated. Chapter 4 presents a study of deuterium exchange on a di-peptide. Deuterium as well as carbon-13 NMR spectroscopy has been extensively used earlier on static powder samples for studying exchange phenomena. In the present study we have applied the methodology for obtaining relative N-D vector orientation in a di-peptide. The magnetization exchange between deuterium nuclei through the dipolar couplings between them has been monitored. The need to match the quadrupolar split energy levels of two different deuterium’s differently oriented in the magnetic field requires that the sample be spun slowly. Characteristic exchange powder patterns were obtained which were used to infer relative orientation information. Comparison with the crystal structure indicates that the magnetization is likely to be inter-molecular rather than intra-molecular. The chapter follows the following sequence. A brief description about the importance of exchange studies in ss-NMR is presented. A theoretical approach is followed by a discussion of the angular dependence of the frequencies and the visualization of the mutual re-orientation angles. The motivation of the project followed by the experimental techniques, especially the use of slow MAS ~ 100 Hz in exchange studies are also presented. Initial studies have been carried out on di-methyl sulphone to check the reproducibility of the earlier reported results and later the sequence is extended to amide proton deuterated di-glycine. The 2D exchange spectrum recorded under slow MAS is then discussed in the context of the crystal structure and possible amide deuteriums involved in the exchange process are inferred. Chapter 5 discusses the natural abundant 13C and 15N NMR spectroscopy in the solid state in designed tri-peptides containing prolines. Proline is a unique amino acid because of it torsion angle values and is responsible for the turns and the globularity of the proteins. The well studied SH3 domain protein often binds to short peptides containing prolines and hence these study gains importance. Three peptides containing prolines were taken up for study. For peptide (1), the conformation was observed as cis/trans in the solution state and for the other two peptides it was all trans. The X-ray studies showed that peptide (1) has two molecules in the unit cell with both cis conformation. This motivated us to look at the solid state spectra of the peptides. Chemical shifts are signatures of conformers and it was established from the chemical shift differences that there exist two molecules in the unit cell for peptide (1), both in cis conformation. The conformers for the other two peptides predicted by NMR chemical shifts also matched with those obtained from X-ray studies. This opens up the possibility of using simple NMR measurements in the solid state as tools for secondary structure determination in larger peptides and proteins.

No description available.

Im Rahmen dieses Promotionsprojektes wurde ein neues, chirales Orientierungsmedium zur Enantiomerenunterscheidung chiraler Amine vorgestellt. Ausgehend von dem enantiomerenreinen (R)- oder (S)-konfigurierten beta-Aminoalkohol, wurde in einer fünfstufigen Synthese (R)- respektive (S)-2-Acrylamido-2-phenylethansulfonsäure ((R)- / (S)-APhES), das zugrundeliegende chirale Monomer dieses neuen Polymergel-basierten Orientierungsmediums, hergestellt. Die daraus resultierenden chiralen Polymergele sind somit in beiden enantiomeren Formen zugänglich. Während die bisher existenten chiralen Orientierungsmedien auf wässrige Lösungen oder apolare organische Lösungsmittel beschränkt waren, zeichnet sich das neue Polymergel durch exzellente Quell- und Orientierungseigenschaften in polaren Lösungsmitteln, wie DMSO, DMF, MeOH und Wasser aus. Im Gegensatz zu den flüssigkristallinen Orientierungsmedien gibt es keine minimale, kritische Konzentration, sodass die Orientierungsstärke, je nach Bedarf, über einen weiten Bereich skaliert werden kann. Neben den exzellenten Orientierungseigenschaften, zeichnet sich dieses neue chirale Polymergel aber hauptsächlich durch seine enantiodiskriminierende Fähigkeit aus. So konnten die beiden Enantiomere des erythro-Mefloquin Hydrochlorids auf Grund unterschiedlicher dipolarer Restkopplungen (RDCs) erfolgreich unterschieden werden. Anhand von Strychnin Hydrochlorid und Menthylamin Hydrochlorid wurde das enantiodiskriminierende Potential der APhES-Gele an zwei weiteren Beispielen erfolgreich demonstriert. In einem unabhängigen Nebenprojekt konnte die absolute Konfiguration von erythro-Mefloquin Hydrochlorid, dem Wirkstoff eines hochrelevanten Anti-Malaria-Medikamentes, erfolgreich bestimmt werden. Bei dem racemisch eingesetzten Wirkstoff wurden seit dessen Entwicklung und besonders im vergangenen Jahrzehnt immer wieder kontroverse Strukturvorschläge bezüglich der absoluten Konfiguration publiziert. Da die beiden etablierten Methoden zur Bestimmung der absoluten Konfiguration, d.h. Totalsynthese oder Kristallstrukturanalyse, zu entgegengesetzten Ergebnissen führten, wurde im Rahmen dieser Promotion eine interdisziplinäre Methode gewählt, bestehend aus den spektroskopischen Verfahren NMR, ORD und ECD in Kombination mit quantenmechanischen DFT-Rechnungen. Basierend auf diesem Ansatz wurde die absolute Konfiguration von (–)-erythro-Mefloquin Hydrochlorid eindeutig als 11R,12S bestimmt. Dieses Ergebnis wurde kurze Zeit später, ebenfalls in dieser Abteilung, durch zwei unabhängige synthetische respektive synthetisch/analytische Methoden bestätigt.

O trocador de Na+/Ca2+ (NCX) constitui um dos principais mecanismos de extrusão de Ca2+ intracelular em células excitáveis. Foi demonstrado que alterações no funcionamento do NCX estão relacionadas a diversas situações patológicas. Por este motivo, o entendimento do mecanismo molecular da manutenção da concentração de Ca2+ intracelular via NCX é importante para a compreensão do funcionamento do trocador, bem como para o desenvolvimento de fármacos. Além de transportar Na+/Ca2+, o NCX também é regulado por esses íons. Este trocador é composto por dois domínios transmembranares, cada um deles contendo 5 &#945;-hélices (TM), e uma grande alça intracelular que conecta as hélices TM5 e TM6. O domínio transmembranar é responsável por catalisar o transporte de Na+/Ca2+ através da bicamada lipídica, enquanto que a alça citoplasmática está envolvida com a regulação do trocador. Esta alça contém dois domínios sensores de Ca2+ adjacentes, denominados CBD1 e CBD2. Apesar da importância fisiológica do NCX, o mecanismo de regulação alostérica do trocador por Ca2+ intracelular permanece desconhecido. Neste trabalho, a espectroscopia de ressonância magnética nuclear (RMN) de alta resolução foi utilizada para investigar a conformação e a dinâmica de CBD1 e CBD2 do trocador de Na+/Ca2+ de Drosophila melanogaster (CALX), isolados ou conectados covalentemente em uma construção denominada CBD12. Um total de 98% das ressonâncias da cadeia principal de CBD1 isolado na presença de Ca2+ foi assinalado, enquanto que na ausência de Ca2+, assinalamentos para apenas uma parte da cadeia principal puderam ser obtidos. Os assinalamentos adquiridos para CBD12 foram baseados na análise de um conjunto de espectros de RMN tridimensional heteronuclear e por comparação com os espectros dos domínios isolados. Uma análise preliminar dos deslocamentos químicos e dos parâmetros de relaxação de 15N obtidos para CBD1 indicou que este domínio é flexível na ausência de Ca2+, mas torna-se rígido após a adição deste íon. As medidas das velocidades de relaxação de 15N e de acoplamentos dipolares residuais (RDCs) de 1H-15N realizadas para CBD12 nas formas apo e holo indicaram que a ligação de Ca2+ em CBD1 estabiliza uma orientação rígida entre os domínios. A análise dos RDCs de 1H-15N mostrou ainda que a orientação média entre CBD1 e CBD2 é praticamente linear na ausência de Ca2+, enquanto que um ângulo menor é assumido após a adição deste íon. Os dados descritos nesta tese suportam um modelo de regulação alostérica em que a modulação da plasticidade de CBD12 pela ligação de Ca2+ no domínio CBD1 controla a abertura e o fechamento do trocador. / The Na+/Ca2+ exchanger (NCX) is a major mechanism for the extrusion of intracellular Ca2+ in excitable cells. It was demonstrated that altered functioning of this protein is related to various pathological situations. Therefore, the understanding of the molecular mechanism for maintaining the intracellular Ca2+ concentration by means of the NCX is important to understand the functioning of the exchanger and to develop drug-based therapies. Besides transporting Na+/Ca2+, the exchanger is also regulated by these ions. The NCX is composed of two transmembrane domains, each of them containing 5 transmembrane alpha-helices (TM), and a very large cytosolic loop that connects TM5 to TM6. The transmembrane domains are responsible for catalyzing the transport of Na+ and Ca2+ ions across the lipid bilayer, while the cytosolic loop is involved in regulation of the exchanger activity. It contains two regulatory Ca2+- binding domains, called CBD1 and CBD2, that appear in tandem. Despite the physiological importance of the NCX, the mechanism of allosteric regulation of the exchanger by intracellular calcium remains unclear. In this work we used high-resolution NMR spectroscopy to study the conformation and the dynamics of the two Ca2+-binding regulatory domains of Drosophila\'s Na+/Ca2+ exchanger (CALX), CBD1 and CBD2, in isolation as well as in a covalent construct called CBD12. Complete backbone NMR resonance assignments were obtained for the isolated CBD1 domain in the Ca2+-bound state, while partial assignments were obtained for CBD1 in the free state. Partial backbone NMR resonance assignments were obtained for the CBD12 construct through the analysis of a standard set of triple resonance NMR spectra. Additional assignments were obtained by comparison with the isolated CBD1 and CBD2 domains. A preliminary analysis of NMR chemical shifts and 15N relaxation data obtained for CBD1 indicates that this domain displays considerable amount of flexibility in the free state, but becomes more rigid upon Ca2+-binding. NMR 15N relaxation rates and 1H-15N residual dipolar couplings (RDCs) obtained for the Apo and Ca2+-bound states of the CBD12 domain indicate that calcium binding stabilizes a rigid inter-domain orientation. Analysis of 1H-15N RDCs further shows that Drosophila\'s CBD12 domain assumes an almost linear inter-domain orientation in the absence of Ca2+, while a smaller inter-domain angle was found in its presence. These findings support a model in which modulation of CBD12 plasticity by the binding of Ca2+ to the CBD1 domain controls the opening and closing of the exchanger.