1 H-detektierte Festkörper-NMR-Studien wasserunzugänglicher Proteine in vitro und in situ

1H-Detektion kann die spektrale Empfindlichkeit in der Festkörper-NMR-Spektroskopie stark verbessern und erlaubt dadurch Studien komplexerer Proteine. Zur 1H-Detektion müssen jedoch austauschbare Wasserprotonen in ansonsten perdeuterierte Proteine eingeführt werden. Diese Vorbedingung hat bisweilen Studien wasserunzugänglicher Proteine stark erschwert. Hier präsentieren wir eine Methode, welche die hochaufgelöste 1H-Detektion wasserunzugänglicher Proteine ermöglicht und selbst in hochkomplexen Medien wie zellulären Oberflächen funktioniert. Wir demonstrieren unsere Methode am Beispiel des K+-Kanals KcsA in Liposomen sowie in situ in nativen bakteriellen Zellmembranen. Wir verwenden unsere Daten für eine Analyse der KcsA-Dynamik und zeigen, dass der Selektivitätsfilter, der in K+-Kanälen hochkonserviert vorliegt und kritisch für die Ionenleitfähigkeit ist, ausgeprägte molekulare Flexibilität aufweist

Deactivation and regeneration of solid acid and base catalyst bodies used in cascade for bio-oil synthesis and upgrading

The modes of deactivation -and the extent to which their properties can be restored- of two catalyst bodies used in cascade for bio-oil synthesis have been studied. These catalysts include a solid acid granulate (namely ZrO2/desilicated zeolite ZSM-5/attapulgite clay) employed in ex-situ catalytic fast pyrolysis of biomass, and a base extrudate (K-exchanged zeolite USY/attapulgite clay) for the subsequent bio-oil upgrading. Post-mortem analyses of both catalyst bodies with Raman spectroscopy and confocal fluorescence microscopy revealed the presence of highly poly-aromatic coke distributed in an egg-shell manner. Deactivation due to coke adsorption onto acid sites affected the zeolite ZSM-5-based catalyst, while for the base catalyst it is structural integrity loss, resulting from KOH-mediated zeolite framework collapse, the main deactivating factor. A hydrothermal regeneration process reversed the detrimental effects of coke in the acid catalyst, largely recovering catalyst acidity (∼80%) and textural properties (∼90%), but worsened the structural damage suffered by the base catalyst

Multiscale Mechanistic Insights of Shaped Catalyst Body Formulations and Their Impact on Catalytic Properties

International audienceZeolite-based catalysts are globally employed in many industrial processes, such as in crude-oil refining and in the production of bulk chemicals. However, to be implemented in industrial reactors efficiently, zeolite powders are required to be shaped in catalyst bodies. Scale-up of zeolite catalysts into such forms comes with side effects to its overall physicochem-ical properties and to those of its constituting components. Although fundamental research into "technical" solid catalysts is scarce, binder effects have been reported to significantly impact their catalytic properties and lifetime. Given the large number of additional (in)organic components added in the formulation, it is somehow surprising to see that there is a distinct lack of research into the unintentional impact organic additives can have on the properties of the zeolite and the catalyst bodies in general. Here, we systematically prepared a series of alumina-bound zeolite ZSM-5-based catalyst bodies, with organic additives such as peptizing, plasticizing, and lubricating agents, to rationalize their impacts on the physicochemical properties of the shaped catalyst bodies. By utilizing a carefully selected arsenal of bulk and high-spatial resolution multiscale characterization techniques, as well as specifically sized bioinspired fluorescent nanoprobes to study pore accessibility, we clearly show that, although the organic additives achieve their primary function of a mechanically robust material, uncontrolled processes are taking place in parallel. We reveal that the extrusion process can lead to zeolite dealumination (from acid peptizing treatment, and localized steaming upon calcination); meso-and macropore structural rearrangement (via burning-out of organic plasticizing and lubricating agents upon calcination); and abating of known alumina binder effects (via scavenging of Al species via chelating lubricating agents), which significantly impact catalytic performance. Understanding the mechanisms behind such effects in industrial-grade catalyst formulations can lead to enhanced design of these important materials, which can improve process efficiency in a vast range of industrial catalytic reactions

Engineering the acidity and accessibility of the zeolite ZSM-5 for efficient bio-oil upgrading in catalytic pyrolysis of lignocellulose

The properties of the zeolite ZSM-5 have been optimised for the production and deoxygenation of the bio-oil∗ (bio-oil on water-free basis) fraction by lignocellulose catalytic pyrolysis. Two ZSM-5 supports possessing high mesopore/external surface area, and therefore enhanced accessibility, have been employed to promote the conversion of the bulky compounds formed in the primary cracking of lignocellulose. These supports are a nanocrystalline material (n-ZSM-5) and a hierarchical sample (h-ZSM-5) of different Si/Al ratios and acid site concentrations. Acidic features of both zeolites have been modified and adjusted by incorporation of ZrO2, which has a significant effect on the concentration and distribution of both Brønsted and Lewis acid sites. These materials have been tested in the catalytic pyrolysis of acid-washed wheat straw (WS-ac) using a two-step (thermal/catalytic) reaction system at different catalyst/biomass ratios. The results obtained have been assessed in terms of oxygen content, energy yield and composition of the produced bio-oil∗, taking also into account the selectivity towards the different deoxygenation pathways. The ZrO2/n-ZSM-5 sample showed remarkable performance in the biomass catalytic pyrolysis, as a result of the appropriate combination of accessibility and acidic properties. In particular, modification of the zeolitic support acidity by incorporation of highly dispersed ZrO2 effectively decreased the extent of secondary reactions, such as severe cracking and coke formation, as well as promoted the conversion of the oligomers formed initially by lignocellulose pyrolysis, thus sharply decreasing the proportion of the components not detected by GC-MS in the upgraded bio-oil∗

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase

Nuclear magnetic resonance (NMR) applied to membrane-protein complexes

Increasing evidence suggests that most proteins occur and function in complexes rather than as isolated entities when embedded in cellular membranes. Nuclear magnetic resonance (NMR) provides increasing possibilities to study structure, dynamics and assembly of such systems. In our review, we discuss recent methodological progress to study membrane-protein complexes (MPCs) by NMR, starting with expression, isotope-labeling and reconstitution protocols. We review approaches to deal with spectral complexity and limited spectral spectroscopic sensitivity that are usually encountered in NMR-based studies of MPCs. We highlight NMR applications in various classes of MPCs, including G-protein-coupled receptors, ion channels and retinal proteins and extend our discussion to protein-protein complexes that span entire cellular compartments or orchestrate processes such as protein transport across or within membranes. These examples demonstrate the growing potential of NMR-based studies of MPCs to provide critical insight into the energetics of protein-ligand and protein-protein interactions that underlie essential biological functions in cellular membranes

Insight into the conformational stability of membrane-embedded BamA using a combined solution and solid-state NMR approach

The β-barrel assembly machinery (BAM) is involved in folding and insertion of outer membrane proteins in Gram-negative bacteria, a process that is still poorly understood. With its 790 residues, BamA presents a challenge to current NMR methods. We utilized a "divide and conquer" approach in which we first obtained resonance assignments for BamA's periplasmic POTRA domains 4 and 5 by solution NMR. Comparison of these assignments to solid-state NMR (ssNMR) data obtained on two BamA constructs including the transmembrane domain and one or two soluble POTRA domains suggested that the fold of POTRA domain 5 critically depends on the interface with POTRA 4. Using specific labeling schemes we furthermore obtained ssNMR resonance assignments for residues in the extracellular loop 6 that is known to be crucial for BamA-mediated substrate folding and insertion. Taken together, our data provide novel insights into the conformational stability of membrane-embedded, non-crystalline BamA