Strukturelle Untersuchung des bakteriellen Amyloids Curli mittels Festkörper-NMR

CsgA is the major subunit of a bacterial functional amyloid called curli, expressed by enterobacteriaceae e.g. Escherichia coli. Curli is involved in formation of biofilms and host cell attachment. Amyloids of CsgA have been extensively studied in vivo as well as in vitro and provide a well-suited system for understanding the process of amyloid formation, propagation and control mechanism. High-resolution structure of functional amyloid fibrils is a prerequisite to gain insights into their functions, mechanism, and more importantly the differences between disease-related and functional amyloids. Solid state NMR was used to investigate CsgA amyloid fibrils. Since spectra of the uniformly labeled CsgA suffered from high spectral overlap due to the high degree of sequence and structure homology in CsgA, segmental labeling by protein-trans-splicing was utilized. Intein fusion pairs for the preparation of three differently labeled samples were established. Solid state NMR measurements on a sample with single labeled structural repeat enabled sequence specific assignments and first insights to the structure of CsgA. The proposed strand-loop-strand motive could be confirmed and distance restraints gave rise to a beta-strand swap within the amyloid fibril. The established strategy of segmental isotope labeling was additionally demonstrated on the structurally well characterized amyloid fibrils of HET-s (218-289). The obtained spectra did not show chemical shift deviations from a uniformly labeled sample and published data. The layer specific labeling pattern allowed the detection on unambiguously intermolecular distance restraints.CsgA ist der Hauptbestandteil eines bakteriellen funktionellen Amyloids, genannt Curli, welches von Enterobakterien wie z.B. Escherichia coli exprimiert wird. Curli ist an der Bildung von Biofilmen und der Bindung an Wirtszellen beteiligt. CsgA amyloide wurden in vivo als auch in vitro ausgiebig untersucht und bilden ein geeignetes System um den Prozess der Amyloidbildung, Ausbreitung und deren Steuermechanismen zu studieren und zu verstehen. Hochaufgelöste Strukturen von Amyloiden sind die Voraussetzung um Einblicke in deren Funktion zu erlangen und vor allem um die Unterschiede zwischen krankheitsbedingten und funktionellen Amyloiden zu verstehen. Dazu wurden CsgA Fibrillen mittels Festkörper-NMR untersucht. Spektren von vollmarkierten Proben (13C, 15N) zeigten eine starke Resonanzüberlagerung aufgrund der intrinsischen Sequenz- und Strukturwiederholungen. Daher wurde eine Methode verwendet in der nur ein bestimmtes Teilstück des Proteins mit Isotopen markiert wird. Erreicht wird dies mit Hilfe von Intein katalysiertem Protein Spleißen. Dazu wurden verschiedene Intein-Fusionsproteine generiert um Proben mit unterschiedlichen Markierungsmustern zu erzeugen. Festkörper-NMR Messungen auf einer Probe mit einer einzelnen markierten strukturellen Wiederholung ermöglichten Sequenz spezifische Resonanzzuordnungen und erste Einblicke in die Struktur von CsgA. Das vermutete Strang-Schleife-Strang Motiv konnte bestätigt werden und Messungen von Abstandsbeschränkungen führten zu der Vermutung eines Beta-Strang-Austausches zwischen zwei aufeinanderfolgenden Molekülen innerhalb der Amyloidfibrillen. Die für CsgA etablierte Strategie der segmentalen Isotopenmarkierung wurde außerdem auf die strukturell gut charakterisierten Amyloidfibrillen von HET-s (218-289) angewandt. Die erhaltenen Spektren zeigten keine Abweichung der chemischen Verschiebung, weder im Vergleich zu einer vollmarkierten Probe noch zu publizierten Daten. Das Ebenen spezifische Markierungsmuster ermöglichte außerdem die Messung von eindeutig intramolekularen Abständen

Strukturelle Untersuchung des bakteriellen Amyloids Curli mittels Festkörper-NMR

CsgA is the major subunit of a bacterial functional amyloid called curli, expressed by enterobacteriaceae e.g. Escherichia coli. Curli is involved in formation of biofilms and host cell attachment. Amyloids of CsgA have been extensively studied in vivo as well as in vitro and provide a well-suited system for understanding the process of amyloid formation, propagation and control mechanism. High-resolution structure of functional amyloid fibrils is a prerequisite to gain insights into their functions, mechanism, and more importantly the differences between disease-related and functional amyloids. Solid state NMR was used to investigate CsgA amyloid fibrils. Since spectra of the uniformly labeled CsgA suffered from high spectral overlap due to the high degree of sequence and structure homology in CsgA, segmental labeling by protein-trans-splicing was utilized. Intein fusion pairs for the preparation of three differently labeled samples were established. Solid state NMR measurements on a sample with single labeled structural repeat enabled sequence specific assignments and first insights to the structure of CsgA. The proposed strand-loop-strand motive could be confirmed and distance restraints gave rise to a beta-strand swap within the amyloid fibril. The established strategy of segmental isotope labeling was additionally demonstrated on the structurally well characterized amyloid fibrils of HET-s (218-289). The obtained spectra did not show chemical shift deviations from a uniformly labeled sample and published data. The layer specific labeling pattern allowed the detection on unambiguously intermolecular distance restraints.CsgA ist der Hauptbestandteil eines bakteriellen funktionellen Amyloids, genannt Curli, welches von Enterobakterien wie z.B. Escherichia coli exprimiert wird. Curli ist an der Bildung von Biofilmen und der Bindung an Wirtszellen beteiligt. CsgA amyloide wurden in vivo als auch in vitro ausgiebig untersucht und bilden ein geeignetes System um den Prozess der Amyloidbildung, Ausbreitung und deren Steuermechanismen zu studieren und zu verstehen. Hochaufgelöste Strukturen von Amyloiden sind die Voraussetzung um Einblicke in deren Funktion zu erlangen und vor allem um die Unterschiede zwischen krankheitsbedingten und funktionellen Amyloiden zu verstehen. Dazu wurden CsgA Fibrillen mittels Festkörper-NMR untersucht. Spektren von vollmarkierten Proben (13C, 15N) zeigten eine starke Resonanzüberlagerung aufgrund der intrinsischen Sequenz- und Strukturwiederholungen. Daher wurde eine Methode verwendet in der nur ein bestimmtes Teilstück des Proteins mit Isotopen markiert wird. Erreicht wird dies mit Hilfe von Intein katalysiertem Protein Spleißen. Dazu wurden verschiedene Intein-Fusionsproteine generiert um Proben mit unterschiedlichen Markierungsmustern zu erzeugen. Festkörper-NMR Messungen auf einer Probe mit einer einzelnen markierten strukturellen Wiederholung ermöglichten Sequenz spezifische Resonanzzuordnungen und erste Einblicke in die Struktur von CsgA. Das vermutete Strang-Schleife-Strang Motiv konnte bestätigt werden und Messungen von Abstandsbeschränkungen führten zu der Vermutung eines Beta-Strang-Austausches zwischen zwei aufeinanderfolgenden Molekülen innerhalb der Amyloidfibrillen. Die für CsgA etablierte Strategie der segmentalen Isotopenmarkierung wurde außerdem auf die strukturell gut charakterisierten Amyloidfibrillen von HET-s (218-289) angewandt. Die erhaltenen Spektren zeigten keine Abweichung der chemischen Verschiebung, weder im Vergleich zu einer vollmarkierten Probe noch zu publizierten Daten. Das Ebenen spezifische Markierungsmuster ermöglichte außerdem die Messung von eindeutig intramolekularen Abständen

Einbau von Rezeptoren in Membran-Nanodisks

In dieser Arbeit wurde sensorisches Rhodopsin II (SRII) und der Photorezeptorkomplex, SRII mit seinem Transducer HtrII in Lipid Nanodisks eingelagert. Nanodisks bestehen aus einer Phospholipid Doppelschicht die von einem Proteinmantel umrandet wird und bilden eine wasserlösliche Modelmembran. Verschiedene biochemische Parameter wurden optimiert um einen möglichst effizienten Einbau der Membranproteine und anschließender Aufreinigung der Disks, zu erreichen. Der Einbau der relevanten Trimer von Dimer Stöchiometrie des SRII/HtrII Photorezeptorkomplex konnte deutlich im Elektronenmikroskop sichtbar gemacht werden. Außerdem wurde durch Messung der Kinetik des Photozyklus des SRII sowie des Komplexes in Nanodisks die Aktivität der Membranproteine nachgewiesen. Abstract: In this work, sensory rhodopsin II (SRII) and the photoreceptor complex, SRII with its transducer HtrII were incorporated into lipid nanodiscs. Nanodiscs consist of a phospholipid bilayer surrounded by a protein shell and form a water-soluble model membrane. Various biochemical parameters were optimized to achieve the most efficient membrane protein reconstitution and subsequent purification of the discs. The incorporation of the relevant trimers of dimers stoichiometry of the SRII/HtrII photoreceptor complex was clearly visualized by electron microscopy. Furthermore, the protein activity was determined by measurements of SRII photocycle in nanodiscs

Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.Peer reviewe

Half a century of amyloids: past, present and future

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward. This journal i

Can Good Institutions Avert the Resource Curse?

To establish the economic impact of petroleum in the UK, this paper uses the synthetic control method. By constructing an artificial UK economy without oil, from the donor pool of OECD economies, it establishes the counterfactual time path of capital stock the UK would have had if it lacked the petroleum endowment. Comparing the observed time path of capital stock with its counterfactual, one can witness that the petroleum extraction has reduced the UK’s capital stock with an average of 17 % since 1970, despite the UK having arguably the best quality of institutions. Dose-response tests suggest that it is possible to attribute the impact to the petroleum production. Performed robustness and sensitivity tests together with several falsification tests show that the result is robust to alternations in the donor pool, the predictor variables and alternative explanations. The finding raises a question regarding the quality of institutions, advanced by Mehlum, Moene and Torvik and claimed that the resource curse only occurs in economies with low quality of institutions

Can Good Institutions Avert the Resource Curse?

To establish the economic impact of petroleum in the UK, this paper uses the synthetic control method. By constructing an artificial UK economy without oil, from the donor pool of OECD economies, it establishes the counterfactual time path of capital stock the UK would have had if it lacked the petroleum endowment. Comparing the observed time path of capital stock with its counterfactual, one can witness that the petroleum extraction has reduced the UK’s capital stock with an average of 17 % since 1970, despite the UK having arguably the best quality of institutions. Dose-response tests suggest that it is possible to attribute the impact to the petroleum production. Performed robustness and sensitivity tests together with several falsification tests show that the result is robust to alternations in the donor pool, the predictor variables and alternative explanations. The finding raises a question regarding the quality of institutions, advanced by Mehlum, Moene and Torvik and claimed that the resource curse only occurs in economies with low quality of institutions

MAS-NMR investigation of the 200 kDa transmembrane channel CorA in lipid bilayers

International audienc

Backbone assignment of crystalline E. coli maltose binding protein

International audienceThe E.coli maltose binding protein (MBP) is a 42.5 kDa molecule widely employed in many biotechnology applications. Because of its molecular size, it has become the main model system for the development of solution NMR methods adapted to large biomolecular targets. Here, we report virtually complete (~90%) backbone resonance assignments obtained on a microcrystalline sample of MBP with 1 H-detected solidstate NMR at fast (>100 kHz) magic-angle spinning. We additionally present the detailed description of the methodology employed for the preparation of the sample and the acquisition and analysis of the NMR spectra. The chemical shifts, obtained with a single uniformly 15 N, 13 C-labelled and fully-protonated sample and about two weeks on a 800 MHz NMR spectrometer, have been deposited to the BMRB under the accession number 50089