Constraining and Modifying Peptides Using Pd-Mediated Cysteine Allylation.

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α3 ß1 (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α3 ß1 .

Nuclear Overhauser spectroscopy in hyperpolarized water - chemical vs. magnetic exchange.

Dissolution dynamic nuclear polarization (dDNP) is a versatile hyperpolarization technique to boost signal intensities in nuclear magnetic resonance (NMR) spectroscopy. The possibility to dissolve biomolecules in a hyperpolarized aqueous buffer under mild conditions has recently widened the scope of NMR by dDNP. The water-to-target hyperpolarization transfer mechanisms remain yet unclear, not least due to an often-encountered dilemma of dDNP experiments: The strongly enhanced signal intensities are accompanied by limited structural information as data acquisition is restricted to short time series of only one-dimensional spectra or a single correlation spectrum. Tackling this challenge, we combine dDNP with molecular dynamics (MD) simulations and predictions of cross-relaxation rates to unravel the spin dynamics of magnetization flow in hyperpolarized solutions.

Identification of the Allosteric Binding Site for Thiazolopyrimidine on the C-Type Lectin Langerin.

Langerin is a mammalian C-type lectin expressed on Langerhans cells in the skin. As an innate immune cell receptor, Langerin is involved in coordinating innate and adaptive immune responses against various incoming threats. We have previously reported a series of thiazolopyrimidines as murine Langerin ligands. Prompted by the observation that its human homologue exhibits different binding specificities for these small molecules, we report here our investigations to define their exact binding site. By using structural comparison and molecular dynamics simulations, we showed that the nonconserved short loops have a high degree of conformational flexibility between the human and murine homologues. Sequence analysis and mutational studies indicated that a pair of residues are essential for the recognition of the thiazolopyrimidines. Taking solvent paramagnetic relaxation enhancement NMR studies together with a series of peptides occupying the same site, we could define the cleft between the short and long loops as the allosteric binding site for these aromatic heterocycles.

Toward protein NMR at physiological concentrations by hyperpolarized water-Finding and mapping uncharted conformational spaces.

Nuclear magnetic resonance (NMR) spectroscopy is a key method for determining the structural dynamics of proteins in their native solution state. However, the low sensitivity of NMR typically necessitates nonphysiologically high sample concentrations, which often limit the relevance of the recorded data. We show how to use hyperpolarized water by dissolution dynamic nuclear polarization (DDNP) to acquire protein spectra at concentrations of 1 µM within seconds and with a high signal-to-noise ratio. The importance of approaching physiological concentrations is demonstrated for the vital MYC-associated factor X, which we show to switch conformations when diluted. While in vitro conditions lead to a population of the well-documented dimer, concentrations lowered by more than two orders of magnitude entail dimer dissociation and formation of a globularly folded monomer. We identified this structure by integrating DDNP with computational techniques to overcome the often-encountered constraint of DDNP of limited structural information provided by the typically detected one-dimensional spectra.

Inversion of Hyperpolarized 13C NMR Signals through Cross-Correlated Cross-Relaxation in Dissolution DNP Experiments.

Dissolution dynamic nuclear polarization (DDNP) is a versatile tool to boost signal amplitudes in solution-state nuclear magnetic resonance (NMR) spectroscopy. For DDNP, nuclei are spin-hyperpolarized "ex situ" in a dedicated DNP device and then transferred to an NMR spectrometer for detection. Dramatic signal enhancements can be achieved, enabling shorter acquisition times, real-time monitoring of fast reactions, and reduced sample concentrations. Here, we show how the sample transfer in DDNP experiments can affect NMR spectra through cross-correlated cross-relaxation (CCR), especially in the case of low-field passages. Such processes can selectively invert signals of 13C spins in proton-carrying moieties. For their investigations, we use schemes for simultaneous or "parallel" detection of hyperpolarized 1H and 13C nuclei. We find that 1H → 13C CCR can invert signals of 13C spins if the proton polarization is close to 100%. We deduce that low-field passage in a DDNP experiment, a common occurrence due to the introduction of so-called "ultra-shielded" magnets, accelerates these effects due to field-dependent paramagnetic relaxation enhancements that can influence CCR. The reported effects are demonstrated for various molecules, laboratory layouts, and DDNP systems. As coupled 13C-1H spin systems are ubiquitous, we expect similar effects to be observed in various DDNP experiments. This might be exploited for selective spectroscopic labeling of hydrocarbons.

Conformational selection of vasopressin upon V1a receptor binding.

The neuropeptide vasopressin (VP) and its three G protein-coupled receptors (V1aR, V1bR and V2R) are of high interest in a wide array of drug discovery programs. V1aR is of particular importance due to its cardiovascular functions and diverse roles in the central nervous system. The structure-activity relationships underpinning ligand-receptor interactions remain however largely unclear, hindering rational drug design. This is not least due to the high structural flexibility of VP in its free as well as receptor-bound states. In this work, we developed a novel approach to reveal features of conformational selectivity upon VP-V1aR complex formation. We employed virtual screening strategies to probe VP's conformational space for transiently adopted structures that favor binding to V1aR. To this end, we dissected the VP conformational space into three sub-ensembles, each containing distinct structural sets for VP's three-residue C-terminal tail. We validated the computational results with experimental nuclear magnetic resonance (NMR) data and docked each sub-ensemble to V1aR. We observed that the conformation of VP's three-residue tail significantly modulated the complex dissociation constants. Solvent-exposed and proline trans-configured VP tail conformations bound to the receptor with three-fold enhanced affinities compared to compacted or cis-configured conformations. The solvent-exposed and more flexible structures facilitated unique interaction patterns between VP and V1aR transmembrane helices 3, 4, and 6 which led to high binding energies. The presented "virtual conformational space screening" approach, integrated with NMR spectroscopy, thus enabled identification and characterization of a conformational selection-type complex formation mechanism that confers novel perspectives on targeting the VP-V1aR interactions at the level of the encounter complex - an aspect that opens novel research avenues for understanding the functionality of the evolutionary selected conformational properties of VP, as well as guidance for ligand design strategies to provide more potent and selective VP analogues.

A novel sample handling system for dissolution dynamic nuclear polarization experiments.

We present a system for facilitated sample vitrification, melting, and transfer in dissolution dynamic nuclear polarization (DDNP) experiments. In DDNP, a sample is typically hyperpolarized at cryogenic temperatures before dissolution with hot solvent and transfer to a nuclear magnetic resonance (NMR) spectrometer for detection in the liquid state. The resulting signal enhancements can exceed 4 orders of magnitude. However, the sudden temperature jump from cryogenic temperatures close to 1 K to ambient conditions imposes a particular challenge. It is necessary to rapidly melt the sample to avoid a prohibitively fast decay of hyperpolarization. Here, we demonstrate a sample dissolution method that facilitates the temperature jump by eliminating the need to open the cryostat used to cool the sample. This is achieved by inserting the sample through an airlock in combination with a dedicated dissolution system that is inserted through the same airlock shortly before the melting event. The advantages are threefold: (1) the cryostat can be operated continuously at low temperatures. (2) The melting process is rapid as no pressurization steps of the cryostat are required. (3) Blockages of the dissolution system due to freezing of solvents during melting and transfer are minimized.