Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Machine Learning Aids Classification and Discrimination of Noncanonical DNA Folding Motifs by an Arrayed Host:Guest Sensing System.

An arrayed host:guest fluorescence sensor system can discriminate among and classify multiple different noncanonical DNA structures by exploiting selective molecular recognition. The sensor is highly selective and can discriminate between folds as similar as native G-quadruplexes and those with bulges or vacancies. The host and guest can form heteroternary complexes with DNA strands, with the host acting as mediator between the DNA and dye, modulating the emission. By applying machine learning algorithms to the sensing data, prediction of the folding state of unknown DNA strands is possible with high fidelity.

Selective Array-Based Sensing of Anabolic Steroids in Aqueous Solution by Host-Guest Reporter Complexes.

Arrayed complexes of a water-soluble deep cavitand and two fluorescent indicators show selective sensing of anabolic-androgenic steroids in aqueous environments. By combining the host-guest complexes with small amounts of heavy metal ions, discrimination between steroids that vary in structure by only a single π bond is possible. The sensing occurs through a triggered aggregation mechanism, which can be mediated by both the presence of metal ions and the steroids. The use of both "turn-on" and "turn-off" fluorophores is essential for good discrimination. As low as 10 µm steroid can be detected, and the discrimination is selective in steroid samples spiked into human urine.

Selective Sensing of Phosphorylated Peptides and Monitoring Kinase and Phosphatase Activity with a Supramolecular Tandem Assay.

Simple tuning of a host:guest pair allows selective sensing of different peptide modifications, exploiting orthogonal recognition mechanisms. Excellent selectivity for either lysine trimethylations or alcohol phosphorylations is possible by simply varying the fluorophore guest. The phosphorylation sensor can be modulated by the presence of small (µM) concentrations of metal ions, allowing array-based sensing. Phosphorylation at serine, threonine, and tyrosine can be selectively sensed via discriminant analysis. The phosphopeptide sensing is effective in the presence of small-molecule phosphates such as ATP, which in turn enables the sensor to be employed in continuous optical assays of both serine kinase and tyrosine phosphatase activity. The activity of multiple different kinases can be monitored, and the sensor is capable of detecting the phosphorylation of peptides containing multiple different modifications, including lysine methylations and acetylation. A single deep cavitand can be used as a "one size fits all" sensor that can selectively detect multiple different modifications to oligopeptides, as well as monitoring the function of their post-translational modification writer and eraser enzymes in complex systems.

Site-Selective Sensing of Histone Methylation Enzyme Activity via an Arrayed Supramolecular Tandem Assay.

Arrayed deep cavitands can be coupled to a fluorescence-based supramolecular tandem assay that allows site-selective in situ monitoring of post-translational modifications catalyzed by the lysine methyltransferase PRDM9 or the lysine demethylase JMJD2E. An arrayed sensor system containing only three cavitand components can detect the specific substrates of enzyme modification, in the presence of other histone peptides in the enzyme assay, enabling investigation of cross-reactivity over multiple methylation sites and interference from nonsubstrate peptides.

Selective Heavy Element Sensing with a Simple Host-Guest Fluorescent Array.

A simple three component array of host-fluorophore complexes is capable of sensitive and selective discrimination of heavy metal ions, including lanthanide and actinide salts in aqueous solution. Instead of applying optical sensors that only use "single-mode" detection, i.e., coordination of the metal to a specific ligand and monitoring the change in emission of an appended fluorophore, we exploit a series of host-fluorophore complexes that are affected by the presence of small amounts of metal ions in aqueous solution in different ways. Variable host-metal and host-guest-metal interactions lead to both turn-on and turn-off fluorescence sensing mechanisms, enhancing the discriminatory properties of the array. The limit of detection for certain metals is as low as 70 nM, and highly similar metals such as lanthanides and actinides can be easily distinguished at low micromolar concentrations in complex salt mixtures.

Enantioselective sensing of insect pheromones in water.

An arrayed combination of water-soluble deep cavitands and cationic dyes has been shown to optically sense insect pheromones at micromolar concentration in water. Machine learning approaches were used to optimize the most effective array components, which allows differentiation between small structural differences in targets, including between different diastereomers, even though the pheromones have no innate chromophore. When combined with chiral additives, enantiodiscrimination is possible, dependent on the size and shape of the pheromone.

Selective discrimination and classification of G-quadruplex structures with a host-guest sensing array.

The secondary structures of nucleic acids have an important influence on their cellular functions but can be difficult to identify and classify quickly. Here, we show that an arrayed suite of synthetic hosts and dyes is capable of fluorescence detection of oligonucleotide secondary structures. Multivariate analysis of different fluorescence enhancements-generated using cationic dyes that show affinity for both DNA G-quadruplexes and the synthetic hosts-enables discrimination between G-quadruplex structures of identical length and highly similar topological types. Different G-quadruplexes that display the same folding topology can also be easily differentiated by the number of G-quartets and sequence differences at the 3' or 5' ends. The array is capable of both differentiation and classification of the G-quadruplex structures at the same time. This simple non-invasive sensing method does not require the discovery and synthesis of specific G-quadruplex binding ligands, but employs a simple multicomponent approach to ensure wide applicability.

Selective sensing of THC and related metabolites in biofluids by host:guest arrays.

A water-soluble host molecule can bind tetrahydrocannabinol (Δ9-THC) and its metabolites in aqueous solution. By pairing this recognition event in a sensing array with fluorescent reporters and varying external mediators, pattern recognition-based detection is possible, which allows selective discrimination of the THC metabolites. The selective sensing can be performed in aqueous solution with micromolar sensitivity, as well as in biofluids such as urine and saliva. Metabolites as similar as Δ8- and Δ9-THC, differing only in the position of a double bond, can be distinguished.

Bridging photochemistry and photomechanics with NMR crystallography: the molecular basis for the macroscopic expansion of an anthracene ester nanorod.

Crystals composed of photoreactive molecules represent a new class of photomechanical materials with the potential to generate large forces on fast timescales. An example is the photodimerization of 9-tert-butyl-anthracene ester (9TBAE) in molecular crystal nanorods that leads to an average elongation of 8%. Previous work showed that this expansion results from the formation of a metastable crystalline product. In this article, it is shown how a novel combination of ensemble oriented-crystal solid-state NMR, X-ray diffraction, and first principles computational modeling can be used to establish the absolute unit cell orientations relative to the shape change, revealing the atomic-resolution mechanism for the photomechanical response and enabling the construction of a model that predicts an elongation of 7.4%, in good agreement with the experimental value. According to this model, the nanorod expansion does not result from an overall change in the volume of the unit cell, but rather from an anisotropic rearrangement of the molecular contents. The ability to understand quantitatively how molecular-level photochemistry generates mechanical displacements allows us to predict that the expansion could be tuned from +9% to -9.5% by controlling the initial orientation of the unit cell with respect to the nanorod axis. This application of NMR-assisted crystallography provides a new tool capable of tying the atomic-level structural rearrangement of the reacting molecular species to the mechanical response of a nanostructured sample.

A supramolecular sensor array for selective immunoglobulin deficiency analysis.

A host-guest based fluorescence sensor array can sense small differences in protein structure. The combination of three cavitand hosts and two fluorophores to form a 4-component array is sufficient to fully discriminate five structurally similar Ig protein isotypes. The array can be applied to recognize Ig deficiencies in serum, when combined with a Protein L-based extraction process, allowing analysis of immunodeficiency in a simpler, lower cost manner than tests that require multiple specific antibodies.

Sensing of citrulline modifications in histone peptides by deep cavitand hosts.

Arrayed cavitand:fluorophore sensor complexes can selectively sense small citrulline modifications at arginine residues on post-translationally modified peptides. The sensor can differentiate between different numbers of citrulline modifications, and a simple two-fluorophore, 6-component array can effect cross-reactive discrimination between single modifications in aqueous solution.

Metal-assisted selective recognition of biothiols by a synthetic receptor array.

A synergistic combination of a deep cavitand host, fluorophore guests and transition metal ions can be used to sense small molecule thiols of biological interest with good efficiency and selectivity in complex aqueous media.

Site selective reading of epigenetic markers by a dual-mode synthetic receptor array.

Variably functionalized self-folding deep cavitands form an arrayed, fluorescent indicator displacement assay system for the detection of post-translationally modified (PTM) histone peptides. The hosts bind trimethyllysine (KMe3) groups, and use secondary upper rim interactions to provide more sensitive discrimination between targets with identical KMe3 binding handles. The sensor array uses multiple different recognition modes to distinguish between miniscule differences in target, such as identical lysine modifications at different sites of histone peptides. In addition, the sensor is affected by global changes in structure, so it is capable of discriminating between identical PTMs, at identical positions on amino acid fragments that vary only in peptide backbone length, and can be applied to detect non-methylation modifications such as acetylation and phosphorylations located multiple residues away from the targeted binding site. The synergistic application of multiple variables allows dual-mode deep cavitands to approach levels of recognition selectivity usually only seen with antibodies.