Literatura académica sobre el tema "Cellular solid-state NMR"

The three-dimensional structures of proteins are among the most valuable contributions of biophysics to the understanding of biological systems (Dickerson & Geis, 1969; Creighton, 1983). Protein structures are utilized in the description and interpretation of a wide variety of biological phenomena, including genetic regulation, enzyme mechanisms, antibody recognition, cellular energetics, and macroscopic mechanical and structural properties of molecular assemblies. Virtually all of the information currently available about the structures of proteins at atomic resolution has been obtained from diffraction studies of single crystals of proteins (Wyckoff et al, 1985). However, recently developed NMR methods are capable of determining the structures of proteins and are now being applied to a variety of systems, including proteins in solution and other non-crystalline environments that are not amenable for X-ray diffraction studies. Solid-state NMR methods are useful for proteins that undergo limited overall reorientation by virtue of their being in the crystalline solid state or integral parts of supramolecular structures that do not reorient rapidly in solution. For reviews of applications of solid-state NMR spectroscopy to biological systems see Torchia and VanderHart (1979), Griffin (1981), Oldfield et al. (1982), Opella (1982), Torchia (1982), Gauesh (1984), Torchia (1984) and Opella (1986). This review describes how solid-state NMR can be used to obtain structural information about proteins. Methods applicable to samples with macroscopic orientation are emphasized.

Abstract Recent advances in the field of in-cell NMR spectroscopy have made it possible to study proteins in the context of bacterial or mammalian cell extracts or even entire cells. As most mammalian cells are part of a multi-cellular complex, there is a need to develop novel NMR approaches enabling the study of proteins within the complexity of a 3D cellular environment. Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using solid-state NMR (ssNMR) spectroscopy. We find that these spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. Finally, we utilize dynamic nuclear polarization (DNP)-supported ssNMR measurements to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids. These findings suggest that solid-state NMR can be used to directly examine proteins or other biomolecules in a 3D cellular microenvironment with potential applications in pharmacological research.

Solid-state nuclear magnetic resonance (ssNMR) is an indispensable tool for elucidating the structure and dynamics of insoluble and non-crystalline biomolecules. The recent advances in the sensitivity-enhancing technique magic-angle spinning dynamic nuclear polarization (MAS-DNP) have substantially expanded the territory of ssNMR investigations and enabled the detection of polymer interfaces in a cellular environment. This article highlights the emerging MAS-DNP approaches and their applications to the analysis of biomolecular composites and intact cells to determine the folding pathway and ligand binding of proteins, the structural polymorphism of low-populated biopolymers, as well as the physical interactions between carbohydrates, proteins, and lignin. These structural features provide an atomic-level understanding of many cellular processes, promoting the development of better biomaterials and inhibitors. It is anticipated that the capabilities of MAS-DNP in biomolecular and biomaterial research will be further enlarged by the rapid development of instrumentation and methodology.

This review summarizes the development of the experimental technique and analytical method for using TD-NMR to study wood-water interactions in recent years. We briefly introduce the general concept of TD-NMR and magnetic resonance imaging (MRI), and demonstrate their applications for characterizing the following aspects of wood-water interactions: water state, fiber saturation state, water distribution at the cellular scale, and water migration in wood. The aim of this review is to provide an overview of the utilizations and future research opportunities of TD-NMR in wood-water relations. It should be noted that this review does not cover the NMR methods that provide chemical resolution of wood macromolecules, such as solid-state NMR.

In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.

Il existe une grande variété de champignons pathogènes humains qui sont à l’origine de maladies bénignes à mortelles. La plupart du temps, ces infections sont associées à d’autres pathologies ou traitements médicaux comme l’asthmes, les leucémies, les transplantations d’organes, le SIDA ou les traitement immunosuppresseurs à base de corticostéroides. Malgré le nombre important de décès et le nombre grandissant d’occurrence des mycoses sévères à travers le monde, les infections fongiques sont encore négligées par les autorités sanitaires.Parmi ces pathogènes fongiques, le champignon filamenteux Aspergillus fumigatus est un des pathogènes principaux du système respiratoire. L’aspergillose, dont les taux de d’infection et de mortalité demeurent élevés, devient un enjeu de santé publique. Les spores d’A. fumigatus sont entourés d’une paroi, essentielle pour leur croissance et leur permettant de résister face au système immunitaire de l’hôte. Cette paroi est composée d’un réseau de polysaccharides recouvert d’un pigment appelé DHN-mélanine et d’une couche de protéines appelées hydrophobines. Ce projet a pour but d’établir l’architecture structurale de la paroi des spores d’A. fumigatus à l’échelle atomique en utilisant la RMN du solide (ssNMR) en rotation à l’angle magique (MAS).D’un autre côté, Cryptococcus neoformans est l’agent pathogène responsable de la cryptococcose ; une mycose affectant le système nerveux central. Cette maladie fongique est, encore de nos jours, une cause significative de mortalité à travers le monde puisqu’elle entraîne de graves symptômes tels que la méningo-encéphalite ; particulièrement fréquente chez les patients déjà infectés par le VIH. C. neoformans se présente sous la forme d’une cellule encapsulée de 5 à 7 μm de diamètre entourée d’une paroi et d’une capsule. Cette paroi, rigide, est liée à la membrane plasmique et composée de polymères d’α-glucan, de β-glucan, de chitine et de chitosan. De plus, la capsule de C. neoformans est majoritairement composée de carbohydrates tels que le glucuronoxylomannan (GXM) (jusqu’à 90 %) ou le glucuronoxylomannogalactan (GXMGal) mais aussi de mannoprotéines et de lipides. Le but de ce projet de thèse est d’identifier les différents composants de la paroi mais aussi de la capsule de C. neoformans par ssNMR et d’établir l’architecture de ces deux entités. Un des aspects de ce projet est aussi d’explorer les possibilités et les limitations des méthodes de détection proton en RMN couplée à un MAS élevé (100 kHz) comme outil d’analyse des parois fongiques.En résumé, puisque la RMN des solides est une méthode de spectroscopie non invasive, nous avons appliqué ce type d’analyses dans le cadre de l’étude de l’architecture moléculaire de systèmes complexes (parois fongiques, capsules, …) dans des conditions aussi proches que possible de l’état natif des cellules. Pendant ces trois années de thèse, nous avons mis en place une méthodologie robuste et rapide permettant d’étudier la composition complexe des structures externes présentes dans les cellules fongiques ainsi que leur architecture au sein des cellules entières. De plus, puisque dans le cadre des infections microbiennes la pathogénicité du microbe repose souvent sur les structures externes des cellules infectieuses, les résultats obtenus au court de cette thèse, apportant une meilleure compréhension de l’organisation cellulaire d’A. fumigatus et C. neoformans, pourraient ainsi être utilisés dans le cadre du développement et de la mise en place de nouvelles stratégies thérapeutiques afin de combattre plus efficacement ces infections fongiques
There is a broad range of fungal pathogen infecting humans and causing diseases that can be from mild to lethal. Severe fungal infections are due to opportunistic pathogens that infect immunosuppressed individuals and are most of the time associated with other diseases or medical conditions such as asthma, leukemia, organ transplants, AIDS or immunosuppressive corticosteroid therapies. Despite the number of deaths and the increase in severe mycosis, fungal infections remain neglected by public health authorities.Among fungal pathogens, the filamentous fungus Aspergillus fumigatus is one of the major pathogen of the respiratory system. Aspergillosis displaying both high incidence and mortality rates, is becoming a massive public health issue. The spores of Aspergillus fumigatus are surrounded by a cell wall, essential for their growth and allowing them to resist against host defense mechanisms. The cell wall is composed of a set of polysaccharides covered by the DHN-melanin pigment and a layer of proteins called hydrophobins. In this project, we aimed at investigated the structural architecture of Aspergillus fumigatus cell wall at atomic resolution using MAS ssNMR spectroscopy.In another hand, Cryptococcus neoformans is the etiological agent of cryptococcosis; which consists in mycosis affecting the central nervous system. This fungal disease remains a significant cause of mortality worldwide by leading to severe symptoms such as meningoencephalitis - especially for immunocompromised individuals suffering from AIDS. C. neoformans results in encapsulated particles with a size of 5-7μm with a two-layers external structure composed of a cell wall and a capsule. The cell wall, rigid, is bounded to the plasma membrane and composed of polymers of α-glucan, β-glucan, chitin and chitosan45. Then, the capsule of C. neoformans is mainly composed of carbohydrates such as glucuronoxylomannan (GXM) (up to 90%), glucuronoxylomannogalactan (GXMGal), mannoproteins and lipids. During this thesis project, we aimed at identifying the different components of C.neoformans cell wall and capsule by ssNMR and to investigate the architecture of these two layers. Part of this project was also the exploration of possibilities and limits of 1H detection methods at fast MAS regime (100 kHz) as the tool to analyze intact cell walls.To sum up, as the solid-state NMR is a non-destructive spectroscopy, we applied this method to the study of the molecular architecture of complex systems (cell wall, capsule…) in cellular conditions – as close as possible to the native state. During these three years, we set up a methodology allowing studying the complex composition of fungal external structures as well as their architecture in the cell context. Finally, because in microbial infections, the pathogenesis often relies on the external structures of the pathogen, all these results could give a better comprehension of the A. fumigatus and C. neoformans cell organization that may help to find new therapeutic strategies to fight, more efficiently, against fungal infections

Indiana University-Purdue University Indianapolis (IUPUI)
Omega-3 polyunsaturated fatty acids (n-3 PUFAs) relieve the symptoms of a wide variety of chronic inflammatory disorders. Typically, they must be obtained in the diet from sources such as fish oils. Docosahexaenoic acid (DHA) is one of these n-3 PUFAs. As yet the structural mechanism responsible for the health benefits within the body is not completely understood. One model that has emerged from biochemical and imaging studies of cells suggests that n-3 PUFAs are taken up into phospholipids in the plasma membrane. Thus the focus here is on the plasma membrane as a site of potential structural modification by DHA. Within cellular membranes, the huge variety of molecules (called lipids) which constitute the membrane suggest inhomogeneous mixing, thus domain formation. One potential domain of interest is called the lipid raft, which is primarily composed of sphingomyelin (SM) and cholesterol (chol). Here the molecular organization of [2H31]-N-palmitoylsphingomyelin (PSM-d31) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane was examined by solid state 2H NMR spectroscopy. Solid state 2H NMR spectroscopy extracts details of molecular orientation and anisotropy of molecular reorientation by analysis of the lineshape. This essentially non-invasive technique allows for a direct measurement of dynamics in bulk materials which has been extensively applied to biological materials. It is a niche area of NMR for which standard software often lack necessary features. Two software programs, “EchoNMR processor” and “EchoNMR simulator”, collectively known as “EchoNMR tools”, that were developed to quickly process and analyze one-dimensional solid-state NMR data, will be described along with some theoretical background of the techniques used. EchoNMR tools has been designed with a focus on usability and the open-source mindset. This is achieved in the in the MATLAB® programming environment which allows for the development of the graphical user interfaces and runs as an interpreter which allows the code to be open-source. The research described here on model membranes demonstrates the utility of the software. The NMR spectra for PSM-d31 in mixtures with PDPC or POPC with cholesterol were interpreted in terms of the presence of nano-sized SM-rich/chol-rich (raft-like) and PC-rich/chol-poor (non-raft) domains that become larger when POPC was replaced by PDPC. An increase in the differential in order and/or thickness between the two types of domains is responsible. The observation of separate signals from PSM-d31, and correspondingly from [3α-2H1]cholesterol (chol-d1) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31), attributed to the raft-like and non-raft domains enabled the determination of the composition of the domains. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain. There was also a substantial amount of PDPC (70%) in the raft-like domain that appears to have minimal effect on the order of SM. PDPC molecules sequestering into small groups to minimize the contact of DHA chains with cholesterol is one possible explanation that would also have implications on raft continuity. These results refine the understanding of how DHA may modulate the structure of raft domains in membranes.

NMR spectroscopy is offering increasing possibilities to obtain structural and dynamic information about macromolecules at atomic resolution. In recent years, it has been extended to the investigation of biological macromolecules in their physiological environment. In-cell NMR spectroscopy allows obtaining physiologically relevant structural and functional information inside living cells through the direct observation of several processes such as protein folding and interaction, metal ion binding, and drugs screening. This thesis aims to widen the application of in-cell NMR for the characterization of the structural and functional properties of proteins as well as their interactions. In a first study, we investigated the folding and the redox state of three human disulphidecontaining proteins (Mia40, Cox17, and SOD1) in the cytoplasm of human and bacterial cells. We successfully determined their redox-state distribution in isolation and with cofactors or redox partners, and found that it is controlled by specific proteins and pathways. In a second study, we employed in vitro and in-cell NMR to characterize the effect of a potential drug (ebselen) on SOD1 mutants. The results revealed that ebselen promotes the correct folding of destabilized SOD1 mutants in cells, and restores their dimerization towards the proper maturation pathway in vitro. Finally, we worked on sample preparation to increase the range of applications of in-cell NMR. On the one hand, we studied several protein systems (CytC, PFN1 and MNK6) in bacterial cells through MAS solid state NMR in order to detect intracellular soluble proteins that are not detectable with canonical experiments of solution NMR. On the other hand, we sought to expand the existing methods of solution in-cell NMR in order to study protein-protein interactions in human cells. In particular, we worked to combine DNA transfection and the delivery of an isotope-labelled recombinant protein to maximize the selectivity of protein labelling inside cells, and minimize the signals of cellular background in the NMR spectra.

Structural biology relies on detailed descriptions of the three-dimensional structures of peptides, proteins, and other biopolymers to explain the form and function of biological systems ranging in complexity from individual molecules to entire organisms. NMR spectroscopy and X-ray crystallography, in combination with several types of calculations, provide the required structural information. In recent years, the structures of several hundred proteins have been determined by one or both of these experimental methods. However, since the protein molecules must either reorient rapidly in samples for multidimensional solution NMR spectroscopy or form high quality single crystals in samples for X-ray crystallography, nearly all of the structures determined up to now have been of the soluble, globular proteins that are found in the cytoplasm and periplasmof cells and fortuitously have these favorable properties. Since only a minority of biological properties are expressed by globular proteins, and proteins, in general, have evolved in order to express specific functions rather than act as samples for experimental studies, there are other classes of proteins whose structures are currently unknown but are of keen interest in structural biology. More than half of all proteins appear to be associated with membranes, and many cellular functions are expressed by proteins in other types of supramolecular complexes with nucleic acids, carbohydrates, or other proteins. The interest in the structures of membrane proteins, structural proteins, and proteins in complexes provides many opportunities for the further development and application of NMR spectroscopy. Our understanding of polypeptides associated with lipids in membranes, in particular, is primitive, especially compared to that for globular proteins. This is largely a consequence of the experimental difficulties encountered in their study by conventional NMR and X-ray approaches. Fortunately, the principal features of two major classes of membrane proteins have been identified from studies of several tractable examples. Bacteriorhodopsin (Henderson et al., 1990), the subunits of the photosynthetic reaction center (Deisenhofer et al., 1985), and filamentous bacteriophage coat proteins (Shon et al., 1991; McDonnell et al., 1993) have all been shown to have long transmembrane hydrophobic helices, shorter amphipathic bridging helices in the plane of the bilayers, both structured and mobile loops connecting the helices, and mobile N- and C-terminal regions.