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Um sich an ändernde Umwelteinflüsse und metabolische Bedürfnisse anpassen zu können, ist es für Zellen essenziell, dass Boten-RNA (engl. messenger RNA, mRNA) stetig und schnell nach der Translation abgebaut wird. In Prokaryoten ist dafür der Proteinkomplex Degradosom verantwortlich, in dem Endo- und Exoribonukleasen RNase E und PNPase das RNA-Transkript in kleinere Fragmente und schließlich einzelne Nukleotide spalten. Die DEAD-Box Helikase RhlB im Komplex dient zusätzlich dazu, mögliche Sekundärstrukturen in der RNA zu entfalten, welche sonst die weitere Degradation behindern würden. Es konnte gezeigt werden, dass RhlB’s sehr geringe katalytische Aktivität – gemessen durch ATP-Verbrauch und Rate an entwundener RNA – signifikant durch die allosterische Bindung an Komplexpartner RNase E erhöht wird. Gleichzeitig deuten andere Studien darauf hin, dass RhlB eine mögliche Selektivität für doppelsträngige RNA-Substrate mit 5‘-Einzelstrang-Überhängen aufweist.
Diese Arbeit liefert neue Erkenntnisse in Bezug auf die Kommunikation zwischen den Degradosom-Komponenten RhlB und RNase E aus E. coli, indem das potenzielle Wechselspiel zwischen RhlBs RNA-Selektivität und der allosterischen Aktivierung durch RNase E untersucht wurde. Der vielseitige Einsatz NMR-spektroskopischer Techniken sowie die Verwendung kurzer RNA-Substrate mit spezifischen Strang-Eigenschaften ermöglicht es, mit einen ungewöhnlichen, RNA-zentrierten Ansatz an diese unzureichend verstandene Protein-Interaktion heranzugehen.
Zunächst wurden hierzu eine Reihe kurzer doppelsträngiger RNA-Konstrukte hergestellt, die sich nicht nur in ihren Einzelstrang-Merkmalen unterscheiden, sondern auch die thermodynamischen Anforderungen eines DEAD-Box Helikase Substrats erfüllen, und gleichzeitig eine ausreichende NMR-spektroskopische Signal-Zuordnung erlauben. Die thermale Stabilität, das Faltungsverhalten sowie die 1H Imino-protonen- und 13C HSQC-Zuordnungen aller geeigneten Konstrukte wurden erfolgreich bestimmt.
Um den Einfluss spezifischer RNA-Substrate sowie die Bindung zweier verschiedener RNase E Fragmente auf RhlBs ATP-Umsatzrate zu untersuchen, wurde sich zunächst eines photometrischen Phosphat-Assays bedient. Damit konnte deutlich gezeigt werden, dass RhlB in Abwesenheit des Komplex-Partners nicht in der Lage ist, signifikante Mengen an ATP umzusetzen, unabhängig davon, welches RNA-Konstrukt eingesetzt wird. Die Bindung der RNase E Fragmente erhöhte signifikant die ATP-Hydrolyse-Rate der Helikase, wobei die größte Aktivierung für den RNA-Duplex mit 5‘-Einzelstrang sowie ein einzelsträngiges Substrat zu beobachten ist. Da diese Ergebnisse deutlich eine RNA-Abhängigkeit beim ATP-Umsatz der Helikase zeigen, wurde untersucht, ob diese Unterschiede ihren Ursprung bereits in der Bindung der spezifischen RNA-Substrate haben. Mittels einer Mischapparatur, die es erlaubt die enzymatische Reaktion direkt im Spektrometer zu initiieren sowie zeitaufgelöster 31P NMR-Experimente konnte die allosterische Aktivierung der ATP-Hydrolyse-Rate von RhlB auch unter NMR-spektroskopischen Messbedingungen nachgewiesen werden.
Da die Ergebnisse des ATPase Assays deutlich eine RNA-Abhängigkeit bei der ATP-Umsatz-Rate der Helikase zeigen, wurde zusätzlich untersucht, ob diese Unterschiede ihren Ursprung in den Affinitäten für die verschiedenen RNA-Substrate haben und ob diese durch die Bindung von RNase E and RhlB beeinflusst werden. Um im gleichen Zuge zu überprüfen, ob die Bindung der RNA an RhlB die RNA-Konformation oder Basenpaarung ändert, werden 1H NMR-Titrationsexperimente durchgeführt. Es konnte erstmals gezeigt werden, dass RhlB eine inhärente Präferenz für Duplexe mit 5‘-Überhang gegenüber Konstrukten mit 3‘-Überhang oder stumpfen Enden besitzt, was sich in einer erhöhten Affinität zeigt. Zusätzlich offenbaren die Messungen, dass RNase Es allosterische Bindung selektiv die Affinität gegenüber Konstrukten mit Einzelstrang-Überhang erhöht, während die Affinität zu RNA Duplexen ohne Überhang sogar verringert wird. Diese Ergebnisse liefern erstmals einen Nachweis, dass RNase E aktiv Einfluss auf RhlBs RNA-Bindung nimmt. Weder die Bindung der RNA and RhlB noch an den RhlB/RNase E Komplex scheint die Basenpaarung oder Konformation der RNA-Substrate zu beeinflussen, da lediglich eine homogene Peak-Verbreitung aller Imino-Protonen-Signale im 1H NMR-Spektrum beobachtet werden konnte.
During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self-replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel (Science 2000, 289, 448–452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self-cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in-line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure-function relation of ribozyme function in this neutral network.
Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding.
Polymorphic G-quadruplex (G4) secondary DNA structures have received increasing attention in medicinal chemistry owing to their key involvement in the regulation of the maintenance of genomic stability, telomere length homeostasis and transcription of important proto-oncogenes. Different classes of G4 ligands have been developed for the potential treatment of several human diseases. Among them, the carbazole scaffold with appropriate side chain appendages has attracted much interest for designing G4 ligands. Because of its large and rigid π-conjugation system and ease of functionalization at three different positions, a variety of carbazole derivatives have been synthesized from various natural or synthetic sources for potential applications in G4-based therapeutics and biosensors. Herein, we provide an updated close-up of the literatures on carbazole-based G4 ligands with particular focus given on their detailed binding insights studied by NMR spectroscopy. The structure-activity relationships and the opportunities and challenges of their potential applications as biosensors and therapeutics are also discussed. This review will provide an overall picture of carbazole ligands with remarkable G4 topological preference, fluorescence properties and significant bioactivity; portraying carbazole as a very promising scaffold for assembling G4 ligands with a range of novel functional applications.
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.
The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio-frequency power to lossy aqueous samples, refolding of barstar from its cold-denatured state can be followed by real-time NMR spectroscopy. Since temperature-induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real-time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low-temperature-unfolded state, which can potentially induce multiple folding pathways. The high time resolution real-time 2D-NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state-correlated spectroscopy, an alternative folding pathway circumventing the rate-limiting cis-trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.
The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
Despite the great interest in glycoproteins, structural information reporting on conformation and dynamics of the sugar moieties are limited. We present a new biochemical method to express proteins with glycans that are selectively labeled with NMR‐active nuclei. We report on the incorporation of 13C‐labeled mannose in the C‐mannosylated UNC‐5 thrombospondin repeat. The conformational landscape of the C‐mannose sugar puckers attached to tryptophan residues of UNC‐5 is characterized by interconversion between the canonical 1C4 state and the B03 / 1S3 state. This flexibility may be essential for protein folding and stabilization. We foresee that this versatile tool to produce proteins with selectively labeled C‐mannose can be applied and adjusted to other systems and modifications and potentially paves a way to advance glycoprotein research by unravelling the dynamical and conformational properties of glycan structures and their interactions.