63 research outputs found
Formation of high-valent iron-oxo species in superoxide reductase: characterization by resonance Raman spectroscopy.
International audienceSuperoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K48 and I118, led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H2O2. These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O-O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site
Homogeneous batch micro-crystallization of proteins from ammonium sulfate
The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to a few micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate alpha-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments.Peer reviewe
Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.Fil: von Stetten, David. Technische Universität Berlin; AlemaniaFil: Seibeck, Sven. Freie Universität Berlin.; AlemaniaFil: Michael, Norbert. Freie Universität Berlin.; AlemaniaFil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; AlemaniaFil: Mroginski, Maria Andrea. Technische Universität Berlin; AlemaniaFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; AlemaniaFil: Krauss, Norbert. Freie Universität Berlin.; AlemaniaFil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; AlemaniaFil: Hildebrandt, Peter. Technische Universität Berlin; AlemaniaFil: Borucki, Berthold. Freie Universität Berlin.; AlemaniaFil: Lamparter, Tilman. Freie Universität Berlin.; Alemani
Structural snapshot of a bacterial phytochrome in its functional intermediate state
Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors
Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease
The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics
X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease
The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2
Untersuchung der Chromophorstruktur in pflanzlichen und bakteriellen Phytochromen durch Vergleich von gemessenen und berechneten Raman-Spektren
Phytochrome sind Photorezeptorproteine in Pflanzen, Bakterien und Pilzen, die eine Vielzahl von biologischen Prozessen regulieren, wie z.B. Blühverhalten und die Synthese von Pigmenten. Der Photozyklus von Phytochromen zeichnet sich durch zwei quasi-stabile Zustände aus (Pr und Pfr), zwischen denen durch Belichtung mit hell- beziehungsweise dunkelrotem Licht reversibel hin- und hergeschaltet werden kann. Als Kofaktor ist in Phytochromen ein offenkettiges Tetrapyrrol gebunden. Die Photoisomerisierung dieses Chromophors löst über mehrere Zwischenschritte schließlich Konformationsänderungen des Proteins aus. Sowohl die molekularen Mechanismen des Photozyklus als auch die folgende Signalweiterleitung sind in weiten Teilen noch unverstanden. Die Resonanz-Raman- (RR-) Spektroskopie erlaubt es, subtile strukturelle Veränderungen des Chromophors zu beobachten, wobei die Raman-Banden der Proteinumgebung durch (Prä-)Resonanzverstärkung der Chromophorbanden unterdrückt werden. Die strukturellen änderungen des Chromophors während des Photozyklus führen zu gänzlich unterschiedlichen RR-Spektren der verschiedenen Zustände von Phytochromen. Sowohl der Einbau von isotopenmarkierten Chromophoren als auch der Austausch einzelner Aminosäuren in der Umgebung des Chromophors beeinflusst die RR-Spektren in charakteristischer Weise, was Rückschlüsse auf die Chromophorstruktur zulässt. Zur Unterstützung der Analyse der experimentellen Daten wurden Raman-Spektren von Modellsystemen des gebundenen Chromophors berechnet, die die Zuordnung der Banden in den experimentellen Spektren erleichtern. Die Berechnungen wurden mit Methoden der Dichte-Funktional-Theorie unter Verwendung von übertragbaren Skalierungsfaktoren für die Kraftkonstanten für eine große Zahl unterschiedlicher Tetrapyrrolgeometrien durchgeführt. Vor allem im Vergleich mit experimentellen Spektren von Phytochromaddukten mit isotopenmarkierten Chromophoren erlauben diese berechneten Spektren die eindeutige Zuordnung individueller Raman-Banden. Die RR-Spektren von Phytochromen aus unterschiedlichen Organismen zeigen sehr ähnliche charakteristische Bandenmuster in den jeweiligen Zuständen des Photozyklus (d.h. Pr, Meta, Pfr). Insbesondere ist der Chromophor der Zustände Pr und Pfr in allen nativen Phytochromen vollständig protoniert, allerdings wird während der Pr -> Pfr Phototransformation entweder Ring B oder C zeitweilig deprotoniert. Erstaunlicherweise zeigen die RR-Spektren der Pr-Zustände der meisten mutierten Phytochromvarianten kaum Unterschiede zu den Wildtypspektren, obwohl der Großteil der ausgetauschten Aminosäuren in der unmittelbaren Umgebung des Chromophors liegt. Bei Belichtung sind die meisten der untersuchten Phytochrom-Mutanten allerdings nicht in der Lage, den Photozyklus bis zum Pfr-Zustand zu durchlaufen. Zwar zeigen die RR-Spektren, dass die primären Schritte des Photozyklus bis hin zum Zustand Meta-Rc in all diesen Proteinen immer noch möglich sind, aber der letzte Schritt (Meta-Rc -> Pfr) ist in den meisten der getesteten Mutationen blockiert, vermutlich weil die Wiederaufnahme des im vorherigen Schritt abgegebenen Protons gestört ist. Die RR-Spektren der Zustände Pr und Pfr von verschiedenen Phytochromen deuten darüberhinaus auf eine konformationelle Heterogenität aus zwei (oder mehr) Chromophorgeometrien hin, die sich hinsichtlich der Torsionswinkel der AB- und CD-Methinbrücken unterscheiden. Die Heterogenität der Meta-Intermediate ist aufgrund unterschiedlicher Protonierungszustände zusätzlich erhöht. Da der Chromophor in seiner inaktiven und aktiven Form (Pr und Pfr) jeweils in mehr als einer Konformation vorliegt, kann die Auslösung der funktionell relevanten Proteinstrukturänderung beim übergang zum Pfr-Zustand nicht mit einer spezifischen Chromophorstruktur verknüpft sein. Stattdessen weisen die Experimente darauf hin, dass diese Strukturänderungen durch die transiente Deprotonierung des Chromophors ausgelöst werden. Basierend auf den Untersuchungen in dieser Arbeit kann damit ein präziseres Modell für den Mechanismus des Photozyklus vorgeschlagen werden, nach dem der Chromophor in Phytochromen nicht als Photoschalter sondern als Phototrigger die Konformationsänderungen des Proteins initiiert.Phytochromes are photosensory proteins that regulate various biological processes in plants, bacteria, and fungi, such as, e.g., flowering or the synthesis of pigments. These functions are linked to the photoactivation of phytochrome which is based on the reversible interconversion between two semi-stable states (Pr and Pfr) by absorption of red or far-red light. The chromophore of phytochrome is a linear tetrapyrrole which undergoes a photoisomerization and subsequent relaxation processes that are eventually coupled to large-scale conformational changes of the protein. Neither the molecular details of the photocycle nor the downstream signal transduction processes are currently well understood. Resonance Raman (RR) spectroscopy allows observing subtle changes of the chromophore structure due to the (pre-)resonance enhancement of the Raman bands of the chromophore over the bands of the protein matrix. The structural changes of the chromophore during the photocycle result in different RR spectra of the various states of phytochromes. The RR spectra of phytochromes with isotopically labeled chromophores or with substitutions of specific amino acids near the chromophore show characteristic effects which allow drawing conclusions regarding the chromophore structure. To complement the experimental data, Raman spectra of model compounds resembling the protein-bound chromophore were calculated in order to obtain an assignment of the bands in the experimental spectra. These calculations, which are based on quantum mechanical force fields obtained by density functional theory and corrected by transferable scaling factors, were carried out for a large number of tetrapyrrole geometries. Especially in comparison with experimental spectra of phytochrome adducts with isotopically labeled chromophores, these calculated Raman spectra allow for the unambiguous assignment of individual vibrational bands. The RR spectra of phytochromes from plants and different other organisms show very similar characteristic band patterns in the respective states of the photocycle (i.e., Pr, Meta, Pfr). The chromophore in the Pr and Pfr states is largely protonated in all native phytochromes, but during the Pr -> Pfr transition, the chromophore is transiently deprotonated at ring B or C. Interestingly, the RR spectra of the Pr states of most mutated phytochromes show hardly any differences compared with the spectra of the wild-type proteins, even though the majority of the substituted residues are in the immediate vicinity of the chromophore. Upon illumination, however, most of these mutant phytochromes are unable to complete the photocycle to the Pfr state. The RR spectra show that the initial steps of the photocycle are still possible in all investigated proteins, but the final step (Meta-Rc -> Pfr) is prohibited in most cases, probably because the transient proton transfer is impaired. The RR spectra of the Pr and Pfr states of several phytochromes suggest a conformational heterogeneity of two (or more) chromophore conformations that differ with respect to the dihedral angles of the AB and CD methine bridges, while the Meta states show an even larger heterogeneity due to the additional presence of different protonation states. Since more than one chromophore conformation exists in both the active and inactive form (Pr and Pfr), the induction of the physiologically relevant changes of the protein structure in the Pr -> Pfr transition cannot be linked to a specific chromophore geometry. Instead, the experiments indicate that the transient deprotonation of the chromophore initiates the structural changes of the protein. Based on the results in this work, a detailed model for the mechanism of the photocycle of phytochromes is suggested, where the chromophore induces the conformational changes of the protein rather as a photo-trigger than as a photo-switch
Epithalamia In honorem nuptiarum Natalium Splendore, Virtutum, Ac Morum, Gratia Praestantissimi Viri, Dn. Sigismundi Traneri Augustani Sponsi. Cum ... Virgine Sibylla von Stetten, ... Dn. Davidis von Stetten Inclytae Reip. Augustanae antehac Senatoris, & rei frumentariae Praefecti dignissimi filia, Sponsa
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Investigation of the chromophore structure in plant and bacterial phytochromes by comparison of experimental and calculated Raman spectra vorgelegt vo
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