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Journal ArticleDOI

Biomolecular NMR Spectroscopy

01 Jul 1997-Radiology (Radiological Society of North America)-Vol. 204, Iss: 1, pp 100-100
TL;DR: It is found that the Pleck-DEP fold contains an additional short helix !
Abstract: The pleckstrin DEP domain structure DEP domains are divergent in sequence. Using multiple sequence alignments and phylogenetic tree reconstructions, we found at least six subfamilies of DEP domains. We selected Pleckstrin, the major substrate of PKC in platelets, for further structural research. We found that the Pleck-DEP fold contains an additional short helix !4 inserted in the "4-"5 loop with respect to other DEP structures. This helix exhibits increased backbone mobility, as shown by NMR relaxation measurements, and may be involved in protein-protein interactions. (Civera et al, 2005.)
Citations
More filters
Journal ArticleDOI
TL;DR: The use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on the experience are described.
Abstract: Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool for fragment-based drug discovery over the last two decades. While NMR has been traditionally used to elucidate the three-dimensional structures and dynamics of biomacromolecules and their interactions, it can also be a very valuable tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. Here, we describe the use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on our experience.

168 citations


Cites background from "Biomolecular NMR Spectroscopy"

  • ...Several experimental NMR approaches for screening have been developed allowing hits to be identified for targets that are small, medium, and large in size (Fernandez and Jahnke 2004; Dalvit 2009; Campos-Olivas 2011; Shortridge and Powers 2011)....

    [...]

Journal ArticleDOI
TL;DR: Solid‐state NMR methods tailored to the construction of 3D molecular structure are discussed and a comparative analysis of 13C′, 13Cα, and 13Cβ resonance frequencies suggests that 13C chemical‐shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility.
Abstract: Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of 13C', 13Calpha, and 13Cbeta resonance frequencies suggests that 13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR.

88 citations

BookDOI
01 Jan 2013
TL;DR: This chapter provides an introduction to the structural and physical biology of viruses by including an elementary overview on virions and the structural basis of virus function and brief structure-based general descriptions of the different stages in the virus cycle.
Abstract: Viruses may be regarded as dynamic nucleoprotein assemblies capable of assisted multiplication within cells, and of propagation between cells and organisms. Infectious virus particles (virions) assembled in a host cell are dynamic, generally metastable particles: They are robust enough to protect the viral genome outside the cell, but are also poised to undergo structural changes and execute mechanochemical actions required for infection of other cells. This chapter provides an introduction to the structural and physical biology of viruses by including: (i) an elementary overview on virions and the structural basis of virus function; (ii) a concise summary on basic techniques used in structural or physical virology; (iii) brief structure-based general descriptions of the different stages in the virus cycle, especially those in which virions and/or their components are involved. These contents may facilitate a better understanding of the specialized subjects treated in the rest of the book. This chapter is also intended as a “road map” to help interconnect and integrate in a single picture the different topics described in depth in the 21 monographic chapters in this book.

76 citations

Book ChapterDOI
02 Mar 2012
TL;DR: In this paper, a review of the study of inclusion complexes between drugs and cyclodextrins by different NMR techniques is presented, including simple measures of 1H-NMR spectrum to more sophisticated experiments, e.g. Diffusion Ordered SpectroscopY (DOSY), NOE methods (ROESY), T1 measure and solid NMR by 13C Cross-Polarization Magic Angle Spinning (CPMAS).
Abstract: Cyclodextrins (CDs) are cyclic oligomers of glucopyranose units that play an important role as a host in inclusion complexes, where non-covalent interactions are involved. They have been extensively studied in supramolecular chemistry. Because of its biocompatibility, relatively non-toxicity and relatively low price, CDs have been widely employed for encapsulation of several substances, being used in food, cosmetic and pharmaceutical industries. Nuclear Magnetic Resonance spectroscopy (NMR) is one of the most useful techniques to study interactions of cyclodextrins with guest compounds. It is relatively easy to apply, the experiments are fast and it is the only technique that provides information on the right orientation of the guest molecule inside the cavity and also on other important parameters related to the physico-chemical characteristics of the inclusion complexes. In this review, it will be discussed the study of inclusion complexes between drugs and cyclodextrins by different NMR techniques. Initially, a brief introduction of the properties of cyclodextrins, its importance as innovative drug carrier systems and its applicability is reviewed. Then different NMR techniques used for characterization of inclusion complexes are detailed, with examples studied in our group, which involves since simple measures of 1H-NMR spectrum to more sophisticated experiments, e.g. Diffusion Ordered SpectroscopY (DOSY), NOE methods (ROESY), T1 measure and solid NMR by 13C Cross-Polarization Magic Angle Spinning (CPMAS).

68 citations


Cites background from "Biomolecular NMR Spectroscopy"

  • ...Besides, NOE cross-peaks can be correlated to their respective internuclear distances (Evans, 1995; Pinto et al., 2005)....

    [...]

Journal ArticleDOI
25 Nov 2014-PLOS ONE
TL;DR: The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained, which reinforces the potential use of h UCBP and CM in tissue regeneration and focus the possible use ofhUCBP as a substitute for the FBS used in h MSCs in vitroculture.
Abstract: Human mesenchymal stem cells (hMSCs) from umbilical cord (UC) blood (UCB) and matrix are tested clinically for a variety of pathologies but in vitro expansion using culture media containing fetal bovine serum (FBS) is essential to achieve appropriate cell numbers for clinical use. Human UCB plasma (hUCBP) can be used as a supplement for hMSCs culture, since UCB is rich in soluble growth factors and due to worldwide increased number of cryopreserved UCB units in public and private banks, without the disadvantages listed for FBS. On the other hand, the culture media enriched in growth factors produced by these hMSCs in expansion (Conditioned medium - CM) can be an alternative to hMSCs application. The CM of the hMSCs from the UC might be a better therapeutic option compared to cell transplantation, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allo- or xeno-transplanted cells. These facts drove us to know the detailed composition of the hUCBP and CM, by 1H-NMR and Multiplexing LASER Bead Technology. hUCBP is an adequate alternative for the FBS and the CM and hUCBP are important sources of growth factors, which can be used in MSCs-based therapies. Some of the major proliferative, chemotactic and immunomodulatory soluble factors (TGF-β, G-CSF, GM-CSF, MCP-1, IL-6, IL-8) were detected in high concentrations in CM and even higher in hUCBP. The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained. The data reinforces the potential use of hUCBP and CM in tissue regeneration and focus the possible use of hUCBP as a substitute for the FBS used in hMSCs in vitro culture.

64 citations

References
More filters
Journal ArticleDOI
TL;DR: The use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on the experience are described.
Abstract: Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool for fragment-based drug discovery over the last two decades. While NMR has been traditionally used to elucidate the three-dimensional structures and dynamics of biomacromolecules and their interactions, it can also be a very valuable tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. Here, we describe the use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on our experience.

168 citations

Journal ArticleDOI
TL;DR: Solid‐state NMR methods tailored to the construction of 3D molecular structure are discussed and a comparative analysis of 13C′, 13Cα, and 13Cβ resonance frequencies suggests that 13C chemical‐shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility.
Abstract: Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of 13C', 13Calpha, and 13Cbeta resonance frequencies suggests that 13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR.

88 citations

BookDOI
01 Jan 2013
TL;DR: This chapter provides an introduction to the structural and physical biology of viruses by including an elementary overview on virions and the structural basis of virus function and brief structure-based general descriptions of the different stages in the virus cycle.
Abstract: Viruses may be regarded as dynamic nucleoprotein assemblies capable of assisted multiplication within cells, and of propagation between cells and organisms. Infectious virus particles (virions) assembled in a host cell are dynamic, generally metastable particles: They are robust enough to protect the viral genome outside the cell, but are also poised to undergo structural changes and execute mechanochemical actions required for infection of other cells. This chapter provides an introduction to the structural and physical biology of viruses by including: (i) an elementary overview on virions and the structural basis of virus function; (ii) a concise summary on basic techniques used in structural or physical virology; (iii) brief structure-based general descriptions of the different stages in the virus cycle, especially those in which virions and/or their components are involved. These contents may facilitate a better understanding of the specialized subjects treated in the rest of the book. This chapter is also intended as a “road map” to help interconnect and integrate in a single picture the different topics described in depth in the 21 monographic chapters in this book.

76 citations

Journal ArticleDOI
25 Nov 2014-PLOS ONE
TL;DR: The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained, which reinforces the potential use of h UCBP and CM in tissue regeneration and focus the possible use ofhUCBP as a substitute for the FBS used in h MSCs in vitroculture.
Abstract: Human mesenchymal stem cells (hMSCs) from umbilical cord (UC) blood (UCB) and matrix are tested clinically for a variety of pathologies but in vitro expansion using culture media containing fetal bovine serum (FBS) is essential to achieve appropriate cell numbers for clinical use. Human UCB plasma (hUCBP) can be used as a supplement for hMSCs culture, since UCB is rich in soluble growth factors and due to worldwide increased number of cryopreserved UCB units in public and private banks, without the disadvantages listed for FBS. On the other hand, the culture media enriched in growth factors produced by these hMSCs in expansion (Conditioned medium - CM) can be an alternative to hMSCs application. The CM of the hMSCs from the UC might be a better therapeutic option compared to cell transplantation, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allo- or xeno-transplanted cells. These facts drove us to know the detailed composition of the hUCBP and CM, by 1H-NMR and Multiplexing LASER Bead Technology. hUCBP is an adequate alternative for the FBS and the CM and hUCBP are important sources of growth factors, which can be used in MSCs-based therapies. Some of the major proliferative, chemotactic and immunomodulatory soluble factors (TGF-β, G-CSF, GM-CSF, MCP-1, IL-6, IL-8) were detected in high concentrations in CM and even higher in hUCBP. The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained. The data reinforces the potential use of hUCBP and CM in tissue regeneration and focus the possible use of hUCBP as a substitute for the FBS used in hMSCs in vitro culture.

64 citations

Journal ArticleDOI
TL;DR: Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked‐brick pattern of adherence to epithelial cells, which is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid.
Abstract: Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface.

46 citations