Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy

It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all detectable species. The analysis was carried out using experimental data obtained during aggregation of the 10.4 kDa Crh protein, which has been shown to involve a partially unfolded intermediate state prior to aggregation. Based on a single real-time 2D 13C–13C transition spectrum, kinetic information about the refolding and aggregation step could be extracted. In addition, structural rearrangements associated with refolding are estimated and several different aggregation scenarios were compared to the experimental data

The architecture of the 10-23 DNAzyme and its implications for DNA-mediated catalysis

Funding Information: The authors acknowledge access to the Jülich‐Düsseldorf Biomolecular NMR Center. HG is grateful for computational support and infrastructure provided by the ‘Zentrum für Informations‐ und Medientechnologie’ (ZIM) at the Heinrich Heine University Düsseldorf and the John von Neumann Institute for Computing (NIC) (user ID: HKF7, VSK33). We thank Hannah Rosenbach for providing activity data. This work was supported by the German Research Foundation (DFG) (103/4‐1, ET 103/4‐3, and the Heisenberg grant ET 103/5‐1) to ME, the Volkswagen Foundation to ME and HG (project no. 9B798) and the European Union‘s Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie grant agreement no. 660258 to AV. Open Access funding enabled and organized by Projekt DEAL. Publisher Copyright: © 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.Understanding the molecular features of catalytically active DNA sequences, so-called DNAzymes, is essential not only for our understanding of the fundamental properties of catalytic nucleic acids in general, but may well be the key to unravelling their full potential via tailored modifications. Our recent findings contributed to the endeavour to assemble a mechanistic picture of DNA-mediated catalysis by providing high-resolution structural insights into the 10-23 DNAzyme (Dz) and exposing a complex interplay between the Dz's unique molecular architecture, conformational plasticity, and dynamic modulation by metal ions as central elements of the DNA catalyst. Here, we discuss key features of our findings and compare them to other studies on similar systems.publishersversionpublishe

Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs

Barttin is an accessory subunit of ClC-K chloride channels expressed in the kidney and the inner ear. Main functions of ClC-K/barttin channels are the generation of the cortico-medullary osmotic gradients in the kidney and the endocochlear potential in the inner ear. Mutations in the gene encoding barttin, BSND, result in impaired urinary concentration and sensory deafness. Barttin is predicted to be a two helical integral membrane protein that directly interacts with its ion channel in the membrane bilayer where it stabilizes the channel complex, promotes its incorporation into the surface membrane and leads to channel activation. It therefore is an attractive target to address fundamental questions of intermolecular communication within the membrane. However, so far inherent challenges in protein expression and stabilization prevented comprehensive in vitro studies and structural characterization. Here we demonstrate that cell-free expression enables production of sufficient quantities of an isotope-labeled barttin variant (I72X Barttin, capable to promote surface membrane insertion and channel activation) for NMR-based structural studies. Additionally, we established purification protocols as well as reconstitution strategies in detergent micelles and phospholipid bilayer nanodiscs. Stability, folding, and NMR data quality are reported as well as a suitable assignment strategy, paving the way to its structural characterization

Leading in the digital age: A systematic review on leader traits in the context of e-leadership

Due to the rapid changes in work environments caused by the Covid-19 pandemic, leadership has shifted from face-to-face to virtual contexts. Accordingly, the new challenges require specific e-leader traits. To summarize the divergent scholarly discussion, we conducted a systematic review and identified distal (i.e., personality, cognitive abilities, motives and attitudes, and core beliefs) and proximal (i.e., skills) attributes of e-leaders. Our results show that some traditional leader traits such as technological, communication, motivational, and organizational skills are also important for e-leadership. However, certain traits become increasingly important in virtual contexts: e-leaders need adaptability and risk- taking to deal with constant change, and higher cultural, social, and emotional intelligence to foster collaboration in diversified teams. In addition, digital technologies require particular change management, coaching, and trust-building skills. Our findings contribute to the current discussion on e-leadership and help practitioners train their leaders towards the identified e-leader profile

Simultaneous use of solution, solid-state NMR and X-ray crystallography to study the conformational landscape of the Crh protein during oligomerization and crystallization

We explore, using the Crh protein dimer as a model, how information from solution NMR, solid-state NMR and X-ray crystallography can be combined using structural bioinformatics methods, in order to get insights into the transition from solution to crystal. Using solid-state NMR chemical shifts, we filtered intra-monomer NMR distance restraints in order to keep only the restraints valid in the solid state. These filtered restraints were added to solid-state NMR restraints recorded on the dimer state to sample the conformational landscape explored during the oligomerization process. The use of non-crystallographic symmetries then permitted the extraction of converged conformers subsets. Ensembles of NMR and crystallographic conformers calculated independently display similar variability in monomer orientation, which supports a funnel shape for the conformational space explored during the solution-crystal transition. Insights into alternative conformations possibly sampled during oligomerization were obtained by analyzing the relative orientation of the two monomers, according to the restraint precision. Molecular dynamics simulations of Crh confirmed the tendencies observed in NMR conformers, as a paradoxical increase of the distance between the two β1a strands, when the structure gets closer to the crystallographic structure, and the role of water bridges in this context

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins

Structural insights from lipid-bilayer nanodiscs link α-Synuclein membrane-binding modes to amyloid fibril formation

Peer reviewe

Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples

Charakterisierung von Proteinpräzipitaten, Aggregationskinetik und Membranproteinen mittels Festkörper-NMR

Die vorliegende Arbeit befasst sich mit der Untersuchung nicht löslicher, biomolekularer Systeme mit Hilfe der Kernmagnetischen-Resonanzspektroskopie (NMR). Einen Schwerpunkt bildet die Erkundung neuer Anwendungsgebiete wie die Messung an aus Lösung ausgefallenen Proteinen und die zweidimensionale Festkörper-Echtzeitspektroskopie. Des Weiteren wurden die Grenzen bekannter Anwendungsgebiete, insbesondere im Bereich der Membranproteine, ausgelotet und durch neue Analysemethoden erweitert. Im Einzelnen konnte ein atomares Bild des präzipitierten Zustandes des Crh-Proteins entwickelt werden. Ausgehend von diesen Ergebnissen konnte erstmalig Proteinaggregation, induziert durch eine temperaturbedingte strukturelle Umwandlung des Crh-Präzipitats, in Echtzeit aufgenommen und kinetisch analysiert werden. Im Weiteren wurde das System der zwei Membranproteine SRII und HtrII untersucht. Neben Untersuchungen an isoliertem SRII führten Messungen an dem Proteinkomplex der beiden Proteine zu einem erweiterten Bild der SRII/HtrII-Bindungsfläche. Zusätzlich wurde die Funktionsweise des SRII/HtrII-Komplexes mit Hilfe von Spektren nach Lichtaktivierung untersucht. Abschließend wurde durch Kombination verschiedener Techniken eine strukturelle Untersuchung des Multidomänen-Membranproteins DcuS im besonderen Hinblick auf dessen Funktion vorgenommen. Hierzu wurden erstmalig die spektroskopischen Daten einer nicht löslichen Domäne im Vergleich zu einer computergestützten Strukturvorhersage analysiert. Die erhaltenen Ergebnisse, sowie Informationen vorausgegangener Untersuchungen ermöglichten die Einführung eines konsistenten Modells der Signalweiterleitung. Vergleiche mit ähnlichen Systemen deuten eine Allgemeingültigkeit des vorgeschlagenen Mechanismus an

Atomare Einblicke in die Dynamik der Membransysteme und der Biokatalyse

Structural biology is targeting increasingly complex systems. Emerging fields such as cellular structural biology consequently apply integrative approaches that combine the strengths of different techniques. In this setting it is of fundamental importance that the applied methods focus on their inherent strengths. The unique features of nuclear-magnetic-resonance (NMR) spectroscopy can play a valuable role in this endeavor. In this article, our research focusing on NMR-method development, tailored to the need of modern structural biology, is outlined