Molecular recognition of an odorant by the murine trace amine-associated receptor TAAR7f.

There are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin, dopamine and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein G and bound to the odorant N,N-dimethylcyclohexylamine (DMCHA) to an overall resolution of 2.

ThermoBRET: A Ligand-Engagement Nanoscale Thermostability Assay Applied to GPCRs.

Measurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre-existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence-detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable to high-throughput screening.

Protein Tethering for Single-Molecule Force Spectroscopy.

Molecular manipulation by optical tweezers is a central technique to study the folded states of individual proteins and how they depend on interactions with molecules including DNA, ligands, and other proteins. One of the key challenges of this approach is to stably attach DNA handles in an efficient manner. Here, we provide detailed descriptions of a universal approach to covalently link long DNA tethers of up to 5000 base pairs to proteins with or without native cysteines.

Molecular recognition of an aversive odorant by the murine trace amine-associated receptor TAAR7f.

There are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein G and bound to the odorant N,N-dimethylcyclohexylamine (DMCH) to an overall resolution of 2.

Direct observation of Hsp90-induced compaction in a protein chain.

The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states.

Combined docking and machine learning identify key molecular determinants of ligand pharmacological activity on β2 adrenoceptor.

G protein-coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands that bind them. Ligands exert their action via the interactions in the ligand binding pocket.

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells.

Pharmacological modulation of cannabinoid type 2 receptor (CBR) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CBR signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CBR fluorescent probes, used successfully across applications, species, and cell types.

Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA-protein coupling.

Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins.

Protein Folding Mediated by Trigger Factor and Hsp70: New Insights from Single-Molecule Approaches.

Chaperones assist in protein folding, but what this common phrase means in concrete terms has remained surprisingly poorly understood. We can readily measure chaperone binding to unfolded proteins, but how they bind and affect proteins along folding trajectories has remained obscure. Here we review recent efforts by our labs and others that are beginning to pry into this issue, with a focus on the chaperones trigger factor and Hsp70.

The chaperone toolbox at the single-molecule level: From clamping to confining.

Protein folding is well known to be supervised by a dedicated class of proteins called chaperones. However, the core mode of action of these molecular machines has remained elusive due to several reasons including the promiscuous nature of the interactions between chaperones and their many clients, as well as the dynamics and heterogeneity of chaperone conformations and the folding process itself. While troublesome for traditional bulk techniques, these properties make an excellent case for the use of single-molecule approaches.

Insight into the Supramolecular Architecture of Intact Diatom Biosilica from DNP-Supported Solid-State NMR Spectroscopy.

Diatom biosilica is an inorganic/organic hybrid with interesting properties. The molecular architecture of the organic material at the atomic and nanometer scale has so far remained unknown, in particular for intact biosilica. A DNP-supported ssNMR approach assisted by microscopy, MS, and MD simulations was applied to study the structural organization of intact biosilica.

Biomolecular DNP-Supported NMR Spectroscopy using Site-Directed Spin Labeling.

Dynamic nuclear polarization (DNP) has been shown to greatly enhance spectroscopic sensitivity, creating novel opportunities for NMR studies on complex and large molecular assemblies in life and material sciences. In such applications, however, site-specificity and spectroscopic resolution become critical factors that are usually difficult to control by current DNP-based approaches. We have examined in detail the effect of directly attaching mono- or biradicals to induce local paramagnetic relaxation effects and, at the same time, to produce sizable DNP enhancements.

NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field.

Dynamic nuclear polarization (DNP) has become a powerful method to enhance spectroscopic sensitivity in the context of magnetic resonance imaging and nuclear magnetic resonance spectroscopy. We show that, compared to DNP at lower field (400 MHz/263 GHz), high field DNP (800 MHz/527 GHz) can significantly enhance spectral resolution and allows exploitation of the paramagnetic relaxation properties of DNP polarizing agents as direct structural probes under magic angle spinning conditions. Applied to a membrane-embedded K(+) channel, this approach allowed us to refine the membrane-embedded channel structure and revealed conformational substates that are present during two different stages of the channel gating cycle.

Platinum-promoted Ga/Al₂O₃ as highly active, selective, and stable catalyst for the dehydrogenation of propane.

A novel catalyst material for the selective dehydrogenation of propane is presented. The catalyst consists of 1000 ppm Pt, 3 wt% Ga, and 0.25 wt% K supported on alumina.

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.