The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of beta-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAP Switch ) that can be conjugated to biomolecules using click chemistry. SNAP Switch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAP Switch , we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAP Switch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

Hexaarylbiimidazoles (HABIs) are a promising class of photoswitchable molecule that have received little attention in the literature. Among them, (2,2′-dimethoxydiphenylimidazole)-1,1′-binaphthyl (HABI1) displays unusual negative photochromism and is responsive to green light. This study investigates the potential of HABIs to serve as photo-responsive actuators controlling the structure of lyotropic liquid crystalline (LLC) materials. HABI1 with four methyl chains and HABI2 with four dodecyl chains were synthesized. Time resolved small angle X-ray scattering was used to characterize the potential disruptive effects of HABIs on the nanostructure of LLC systems. HABIs underwent rapid isomerization under irradiation, with a very slow reversion in the dark in toluene and in the LLC matrix, demonstrating excellent stability and photo-fatigue resistant. HABIs completely triggered phase transitions in the phytantriol-based materials, and HABI2 generated a greater disruption than HABI1 on the lipid packing due to the enhanced steric influence. Tuning the lipid composition yielded systems that transitioned from a “slow release” lamellar phase to a “burst release” bicontinuous cubic phase upon light irradiation. Such systems therefore may exhibit a triggered release behavior upon a short time of irradiation, showing great potential in “on demand” drug delivery.

Light-responsive nanocarriers are applicable as non-invasive, highly tunable and precisely controlled drug delivery systems. Here, we report a new nanocarrier system, achieved by doping D1, a type of green light-responsive donor acceptor Stenhouse adduct (DASA), into a lipid-based lyotropic liquid crystalline system. Time-resolved small angle X-ray scattering was used to confirm that the matrix underwent a rapid and fully reversible phase transition from lamellar to inverse cubic phase upon irradiation with green light (532 nm), reverting back on removal of light. Fluorescein isothiocyanate-dextran (FD4) was used as a model hydrophilic cargo. The release of cargo upon varying irradiation parameters was investigated in vitro which showed that irradiation can trigger a burst release of FD4 upon phase transition. This additive shows promise for the development of new visible light-activated, "on demand" drug delivery systems. (C) 2019 Elsevier Inc. All rights reserved.

Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.

Stimuli-responsive nanocarriers can release their bioactive cargo “on demand”, potentially improving the selectivity, specificity, and efficacy of therapeutic drugs. Light of wavelengths between 650–1100 nm is a unique stimulus with many advantages. It is noninvasive and “bio-friendly” and can be manipulated with precision and ease. Many light-responsive nanocarriers reported in the literature employ photochromic molecules that undergo reversible isomerization upon irradiation, thereby functioning as gates or switches for the release of encapsulated bioactive compounds. This review surveys the design, synthesis, function, and application of one-photon visible and near-infrared light-triggered photoswitchable compounds in the development of long-wavelength light-responsive nanocarriers.

Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-μm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.

The C7-Gd and C8-Gd tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the C7-Gd ( S configuration) and C8-Gd ( R configuration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band 1H-ENDOR (electron-electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron-electron resonance) measurements were insensitive to the R or S configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the C7-Gd and C8-Gd tags are attractive Gd(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy.

Photocrosslinkable diacetylenes were used for the biocompatibilization of upconverting nanoparticles (UCNPs). In their Communication on page 16036 ff., T. Joshi, H. Stephan, K. Zarschler, and co-workers demonstrate that biomolecular corona-resistant UCNPs can be generated by first coating the UCNP surface with a mixture of diacetylenic ligands and phospholipids, followed by irradiation with UV light. These ene–yne cross-linked bright UCNPs contain sites for linking bio/radioactive moieties.

The ability to determine the amount of material endocytosed by a cell is important for our understanding of cell biology and in the design of effective carriers for drug delivery. To quantify internalization by fluorescence, the signal from material remaining on the cell surface must be differentiated from endocytosed material. Sensors for internalization offer advantages over traditional methods for achieving this as they exhibit improved sensitivity, allow for multiple fluorescent markers to be used simultaneously, and are amenable to high-throughput analysis. We have developed a small fluorescent internalization sensor, similar in size to a standard fluorescent dye, that can be conjugated to proteins and uses the rapid and highly specific bio-orthogonal reaction between a tetrazine and a trans-cyclooctene group to switch off the surface signal. The sensor can be attached to a variety of materials using simple chemistry and is compatible with flow cytometry and fluorescence microscopy, making it a useful tool to study the uptake of material into cells.

The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p-cyano-phenylalanine. The 1H-NMR signals of the tert-butyl and TMS groups were compared to the 1H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54 kDa. The 1H-NMR resonance of the TMS group appeared near 0 ppm in a spectral region with few protein resonances, facilitating the observation of signal changes in response to ligand binding. In all proteins, the R 2 relaxation rate of the tert-butyl group of Tbf was only little greater than that of Tby (less than two-fold). Deuteration of the phenyl ring of Tbf made only a relatively small difference. The effective T 2 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.

•Recent advances in the field of lanthanide-based chemosensors are reviewed.•Sensors for metal cations, anions and neutral small molecules are discussed.•The design features and mechanisms by which sensors function are emphasised.•Contributions of a number of Australian-based researchers are highlighted.

Protein–ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B–NS3 protease and the human prolyl isomerase FK506 binding protein.

Durch Licht vernetzbare Diacetylene machen aufwärtskonvertierende Nanopartikel (UCNPs) biokompatibel. In ihrer Zuschrift auf S. 16268 ff. demonstrieren T. Joshi, H. Stephan, K. Zarschler et al., dass UCNPs, die keine Biomolekülkorona anlagern, erzeugt werden können, indem die UCNP-Oberfläche zuerst mit einer Mischung von Diacetylenliganden und Phospholipiden überzogen und anschließend mit UV-Licht bestrahlt wird. Die En-In-vernetzten, hellen UCNPs enthalten zudem Anbindungsstellen für bio-/radioaktive Einheiten.

Pure hexagonal (β-phase) NaYF4-based hydrophobic upconverting nanoparticles (UCNPs) were surface-modified with O-phospho-l-threonine (OPLT), alendronic acid, and PEG-phosphate ligands to generate water-dispersible UCNPs. Fourier-transform infrared (FTIR) spectroscopy was used to establish the presence of the ligands on the UCNP surface. These UCNPs exhibit great colloidal stability and a near-neutral surface at physiological pH, as confirmed by dynamic light scattering (DLS) and zeta potential (ζ) measurements, respectively. The particles also display excellent long-term stability, with no major adverse effect on the size of UCNPs when kept at pH 7.4. Upon exposure to human serum, PEG-phosphate- and alendronate-coated UCNPs showed no formation of biomolecular corona, as confirmed by SDS-PAGE analysis. The photophysical properties of water-dispersible UCNPs were investigated using steady-state as well as time-resolved luminescence spectroscopy, under excitation at ca. 800 nm. The results clearly show that the UCNPs demonstrate bright upconversion (UC) luminescence. Furthermore, the presence of reactive groups on the NPs, such as free amine groups in alendronate-coated UCNPs, enables further functionalisation of UCNPs with, for example, small molecules, peptides, proteins, and antibodies. Overall these protein corona resistant UCNPs show great biocompatibility and are worthy of further investigation as potential new biomaging probes.

An in-depth study of the interaction of a trinuclear terbium(III)-dizinc(II) complex with an array of nucleotides differing in the type of nucleobase and number of phosphate groups, as well as cyclic versus acyclic variants, is presented. The study examined the nature of the interaction and the efficiency at which guanine was able to sensitize terbium(III) luminescence. Competitive binding and titration studies were performed to help establish the nature/mode of the interactions. These established that (1) interaction occurs by the coordination of phosphate groups to zinc(II) (in addition to uridine in the case of uridine monophosphate), (2) acyclic nucleotides bind more strongly than cyclic counterparts because of their higher negative charge, (3) guanine-containing nucleotides are able to sensitize terbium(III) luminescence with the efficiency of sensitization following the order guanosine monophosphate (GMP) > guanosine diphosphate > guanosine triphosphate because of the mode of binding, and (4) nucleoside monophosphates bind to a single zinc(II) ion, whereas di- and triphosphates appear to bind in a bridging mode between two host molecules. Furthermore, it has been shown that guanine is a sensitizer of terbium(III) luminescence. On the basis of the ability of GMP to effectively sensitize terbium(III)-based luminescence while cyclic GMP (cGMP) does not, the complex has been utilized to monitor the catalytic conversion of cGMP to GMP by a phosphodiesterase enzyme in real time using time-gated luminescence on a benchtop fluorimeter. The complex has the potential to find broad application in monitoring the activity of enzymes that process nucleotides (co)substrates, including high-throughput drug-screening programs.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.

A method for temperature-dependent sensing of both inorganic mercury (Hg2+) and organic methyl mercury (MeHg+) with the same gold nanoparticle (AuNP) system was achieved. The AuNPs were functionalised with a thymine moiety and aggregation was achieved through binding of a single mercuric species between two thymine molecules on the surface of the nanoparticles. Both Hg2+ and MeHg+ caused a significant change in the visible absorbance spectrum, resulting in a colour change from red to blue. Significantly, the two mercuric species produced different hues of blue, distinguishable by eye. This method could lead to the development of a method for tandem detection of Hg2+ and MeHg+ in environmental samples.