Hiking and digital innovation: Analysis strategies for tourist destinations management

Hiking is a recreational and outdoor activity that could experience significant growth in France with an increasing role in destination attractiveness and development. Having become a fully-fledged territorial marketing tool, hiking comes becomes integral to many destination management strategies. Hiking benefits from a growing social enthusiasm nurtured by the adoption of digital tools. The research question is the following: How does hiking fit into territorial development strategies for tourist destinations that are investing in digital tools? This paper is aims at presenting the latest developments of the research project, the conceptual approach (actor network theory), and the methodology. The results of an exploratory study will be presented, as well as the perspectives they present for the second phase of the research project

Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3 labelling: application to the 50S ribosome subunit

International audienceSolid-state NMR spectroscopy allows the characterization of structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths only marginally increase. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus

Specific isotopic labelling and reverse labelling for protein NMR spectroscopy : using metabolic precursors in sample preparation

The study of protein structure, dynamics and function by NMR spectroscopy commonly requires samples that have been enriched ('labelled') with the stable isotopes 13C and/or 15N. The standard approach is to uniformly label a protein with one or both of these nuclei such that all C and/or N sites are in principle 'NMR-visible'. NMR spectra of uniformly labelled proteins can be highly complicated and suffer from signal overlap. Moreover, as molecular size increases the linewidths of NMR signals broaden, which decreases sensitivity and causes further spectral congestion. Both effects can limit the type and quality of information available from NMR data. Problems associated with signal overlap and signal broadening can often be alleviated though the use of alternative, non-uniform isotopic labelling patterns. Specific isotopic labelling 'turns on' signals at selected sites while the rest of the protein is NMR-invisible. Conversely, specific isotopic unlabelling (also called 'reverse' labelling) 'turns off' selected signals while the rest of the protein remains NMR-visible. Both approaches can simplify NMR spectra, improve sensitivity, facilitate resonance assignment and permit a range of different NMR strategies when combined with other labelling tools and NMR experiments. Here, we review methods for producing proteins with enrichment of stable NMR-visible isotopes, with particular focus on residue-specific labelling and reverse labelling using Escherichia coli expression systems. We also explore how these approaches can aid NMR studies of proteins

Computational Reverse-Engineering of a Spider-Venom Derived Peptide Active Against Plasmodium falciparum SUB1

merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity. culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1.Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering

Caractérisation de l'interaction entre la glycoprotéine d'enveloppe gp120 du VIH-1 et les héparanes sulfate : importance des changements conformationnels induits par la liaison à CD4

During viral entry, HIV surface glycoprotein gp120 binds to CD4, thereby anchoring the virus to the host cell surface. This interaction induces conformational changes within gp120 that expose CD4-induced (CD4i) epitope, the binding site for coreceptors (usually a member of the chemokine receptor family). In addition, HIV binds to heparan sulphate (HS), an abundant cell surface polysaccharide, through several domains of gp120, including CD4i. Thus, it may be possible to inhibit HIV binding to cells, using soluble molecules derived from HS. In this context, we proposed to characterise further the structural features of the CD4i/HS interaction. First, we produced and purified gp120 proteins mutated on four residues within CD4i, suspected to interact with HS and, then, we studied their ability to bind to heparin by SPR. A second aspect of the project concerned the development of a new strategy for defining critical residues involved in protein/heparin interactions. Results led to the identification of four heparin binding domains within gp120 and, in particular, three residues within CD4i (R419, K421 and K432) were reported to bind heparin. These studies should contribute to clarify the role of HS in the mechanism of HIV binding to the cell surface and provide precise structural information enabling the definition of HS-derived inhibitors of viral entry.Lors de l'attachement du VIH à la surface d'une cellule, la protéine d'enveloppe gp120 se fixe au récepteur cellulaire CD4, exposant alors son site CD4i qui est alors reconnu par des corécepteurs. Par ailleurs, Le VIH est capable de se fixer aux héparanes sulfate (HS), des polysaccharides présents en abondance à la surface cellulaire, notamment via le site CD4i de gp120 (site de fixation des corécepteurs). Il serait donc possible d'inhiber l'attachement du VIH sur les cellules par l'utilisation de molécules solubles dérivées des HS. Dans ce contexte, nos travaux se sont attachés à définir les aspects structuraux de l'interaction VIH/HS au niveau du site CD4i. Pour cela, des protéines gp120 mutées sur quatre résidus du site CD4i, potentiellement engagés dans la fixation des HS, ont été produites, purifiées et étudiées, par BIAcore, pour leur capacité à interagir avec l'héparine. En parallèle, nous avons développé une méthode simple, permettant d'identifier les régions de fixation à l'héparine d'une protéine donnée. L'ensemble de ces travaux nous a permis d'identifier, au sein de gp120, quatre domaines de liaison à l'héparine et de valider, en particulier, l'engagement de trois résidus du site CD4i (R419, K421 et K432) dans l'interaction avec le polysaccharide. Ces différentes approches ont pour but de clarifier le rôle des HS dans le processus d'attachement du virus à la surface cellulaire et de fournir des informations structurales précises permettant la définition de composés issus des HS capables d'inhiber le mécanisme de l'entrée virale

L’économisation des loisirs pédestres. Structuration et dynamique du marché sur le littoral

International audienc

The market of coastal hiking. An approach based on “market-agencements”

International audienc

Caractérisation de l interaction entre la glycoprotéine d enveloppe gp120 du VIH-1 et les héparanes sulfate : importance des changements conformationnels induits par la liaison à CD4

Lors de l attachement du VIH à la surface d une cellule, la protéine d enveloppe gp120 se fixe au récepteur cellulaire CD4, exposant alors son site CD4i qui est alors reconnu par des corécepteurs. Par ailleurs, Le VIH est capable de se fixer aux héparanes sulfate (HS), des polysaccharides présents en abondance à la surface cellulaire, notamment via le site CD4i de gp120 (site de fixation des corécepteurs). Il serait donc possible d inhiber l attachement du VIH sur les cellules par l utilisation de molécules solubles dérivées des HS. Dans ce contexte, nos travaux se sont attachés à définir les aspects structuraux de l interaction VIH/HS au niveau du site CD4i. Pour cela, des protéines gp120 mutées sur quatre résidus du site CD4i, potentiellement engagés dans la fixation des HS, ont été produites, purifiées et étudiées, par BIAcore, pour leur capacité à interagir avec l héparine. En parallèle, nous avons développé une méthode simple, permettant d identifier les régions de fixation à l héparine d une protéine donnée. L ensemble de ces travaux nous a permis d identifier, au sein de gp120, quatre domaines de liaison à l héparine et de valider, en particulier, l engagement de trois résidus du site CD4i (R419, K421 et K432) dans l interaction avec le polysaccharide. Ces différentes approches ont pour but de clarifier le rôle des HS dans le processus d attachement du virus à la surface cellulaire et de fournir des informations structurales précises permettant la définition de composés issus des HS capables d inhiber le mécanisme de l entrée virale.During viral entry, HIV surface glycoprotein gp120 binds to CD4, thereby anchoring the virus to the host cell surface. This interaction induces conformational changes within gp120 that expose CD4-induced (CD4i) epitope, the binding site for coreceptors (usually a member of the chemokine receptor family). In addition, HIV binds to heparan sulphate (HS), an abundant cell surface polysaccharide, through several domains of gp120, including CD4i. Thus, it may be possible to inhibit HIV binding to cells, using soluble molecules derived from HS. In this context, we proposed to characterise further the structural features of the CD4i/HS interaction. First, we produced and purified gp120 proteins mutated on four residues within CD4i, suspected to interact with HS and, then, we studied their ability to bind to heparin by SPR. A second aspect of the project concerned the development of a new strategy for defining critical residues involved in protein/heparin interactions. Results led to the identification of four heparin binding domains within gp120 and, in particular, three residues within CD4i (R419, K421 and K432) were reported to bind heparin. These studies should contribute to clarify the role of HS in the mechanism of HIV binding to the cell surface and provide precise structural information enabling the definition of HS-derived inhibitors of viral entry.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

NMR assignment of human HSP90 N-terminal domain bound to a long residence time resorcinol ligand

International audienceHSP90 is a major molecular chaperone that helps both folding and stabilization of various client proteins often implicated in growth control and cell survival such as kinases and transcription factors. However, among HSP90 clients are also found numerous oncoproteins and, through its assistance to them, HSP90 has consequently been reported as a promising anticancer target. Several ligand chemotypes, including resorcinol type ligands, were found to inhibit HSP90, most of them in an ATP competitive manner. Binding of some of these ligands modify significantly the NMR spectrum of the HSP90 ATP binding domain compared to the apo protein spectrum, hampering assignment transfer from the previously assigned human HSP90 apo state. Here we report the assignment of the 1HN, 15N, 13C', 13Cα, 13Cβ, 1Hmethyl, and 13Cmethyl chemical shifts of the 29 kDa HSP90 N-terminal domain bound to a long residence time resorcinol type inhibitor: 5-[4-(2-Fluoro-phenyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]-N-furan-2-ylmethyl-2,4-dihydroxy-N-methyl-benzamide. 92% of the backbone resonances and 100% of the [1H, 13C]-resonances of Aβ, Mε, Tγ, Lδ2, Vγ2 and Iδ1 methyl groups were successfully assigned, including for the first time the assignment of the segment covering the nucleotide/drug binding site. Secondary structure predictions based on the NMR assignment reveal a structural rearrangement of HSP90 N-terminal domain upon ligand binding. The long residence time ligand induces the formation of a continuous helix covering the ligand binding site of HSP90 N-terminal domain accounting for the large differences observed in the NMR spectra between the apo and bound proteins

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

International audienceThe specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies