Wittmann, Valentin
0000-0003-4043-6813 Berufsbeschreibung
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In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code
The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-L-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels–Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins.
Protein Spin Labeling with a Photocaged Nitroxide Using Diels-Alder Chemistry
EPR spectroscopy of diamagnetic bio-macromolecules is based on site-directed spin labeling (SDSL). Here, we present a novel labeling strategy for proteins. We developed and synthesized a nitroxide-based spin label that can be ligated to proteins by an inverse-electron-demand Diels-Alder (DAinv) cycloaddition to genetically encoded non-canonical amino acids (ncAA). The nitroxide moiety is shielded by a photoremovable protecting group (PPG) with an attached tetraethylene glycol unit to achieve water solubility. We demonstrate SDSL of two model proteins with the PaNDA (Photoactivatable Nitroxide for DAinv reaction) label. Our strategy features high reaction rates combined with high selectivity, and the possibility to deprotect the nitroxide in E. coli lysate.
Mechanism of multivalent carbohydrate-protein interactions studied by EPR spectroscopy
From a distance: Distance measurements in the nanometer range by means of spin-label electron paramagnetic resonance provide structural evidence for multivalent protein–ligand interactions in solution (see picture; protein subunits: blue/green, ligand: black, spin labels: yellow circles, binding sites: yellow letters). The data show a detailed picture of the binding of divalent ligands to a lectin.
Precipitation-free high-affinity multivalent binding by inline lectin ligands
Multivalent ligand–protein interactions are a key concept in biology mediating, for example, signalling and adhesion. Multivalent ligands often have tremendously increased binding affinities. However, they also can cause crosslinking of receptor molecules leading to precipitation of ligand–receptor complexes. Plaque formation due to precipitation is a known characteristic of numerous fatal diseases limiting a potential medical application of multivalent ligands with a precipitating binding mode. Here, we present a new design of high-potency multivalent ligands featuring an inline arrangement of ligand epitopes with exceptionally high binding affinities in the low nanomolar range. At the same time, we show with a multi-methodological approach that precipitation of the receptor is prevented. We distinguish distinct binding modes of the ligands, in particular we elucidate a unique chelating binding mode, where four receptor binding sites are simultaneously bridged by one multivalent ligand molecule. The new design concept of inline multivalent ligands, which we established for the well-investigated model lectin wheat germ agglutinin, has great potential for the development of high-potency multivalent inhibitors as future therapeutics.
Conformationally Unambiguous Spin Label for Exploring the Binding Site Topology of Multivalent Systems
Multivalent carbohydrate–lectin interactions are a key concept in biological processes mediating, for example, signaling and adhesion. Binding affinities of multivalent ligands often increase by orders of magnitude compared to a monovalent binding situation. Thus, the design of multivalent ligands as potent inhibitors is a highly active field of research, where knowledge about the binding site topology is crucial. Here, we report a general strategy for precise distance measurements between the binding sites of multivalent target proteins using monovalent ligands. We designed and synthesized Monovalent, conformationally Unambiguously Spin-labeled LIgands (MUeSLI). Distances between the binding sites of the multivalent model lectin wheat germ agglutinin in complex with a GlcNAc-derived MUeSLI were determined using pulsed electron paramagnetic resonance spectroscopy. This approach is an efficient method for exploring multivalent systems with monovalent ligands, and it is readily transferable to other target proteins, allowing the targeted design of multivalent ligands without structural information available.
Mechanistische Untersuchung multivalenter Kohlenhydrat-Protein-Wechselwirkungen durch EPR-Spektroskopie
Durch Abstandsmessungen mit Spinsonden-EPR-Spektroskopie gelang der direkte Nachweis multivalenter Protein-Ligand-Interaktionen in Lösung. Man erhält ein detailliertes Bild des Mechanismus der Bindung divalenter Liganden an ein Lectin. Chelatbindung lässt sich so von monovalentem Binden mehrerer Moleküle unterscheiden.
Optimising broadband pulses for DEER depends on concentration and distance range of interest
EPR distance determination in the nanometre region has become an important tool for studying the structure and interaction of macromolecules. Arbitrary waveform generators (AWGs), which have recently become commercially available for EPR spectrometers, have the potential to increase the sensitivity of the most common technique, double electron–electron resonance (DEER, also called PELDOR), as they allow the generation of broadband pulses. There are several families of broadband pulses, which are different in general pulse shape and the parameters that define them. Here, we compare the most common broadband pulses. When broadband pulses lead to a larger modulation depth, they also increase the background decay of the DEER trace. Depending on the dipolar evolution time, this can significantly increase the noise level towards the end of the form factor and limit the potential increase in the modulation-to-noise ratio (MNR). We found asymmetric hyperbolic secant (HS{1,6}) pulses to perform best for short DEER traces, leading to a MNR improvement of up to 86 % compared to rectangular pulses. For longer traces we found symmetric hyperbolic secant (HS{1,1}) pulses to perform best; however, the increase compared to rectangular pulses goes down to 43 %.