Purification and crystallization of a non-GluR2 AMPA-receptor ligand-binding domain: a case of cryo-incompatibility addressed by room-temperature data collection

Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA-Rs) have also been implicated in a number of neurological disorders. To investigate subunit-specific differences in the ligand binding and activation of AMPA-Rs, the GluR4 AMPA-R ligand-binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non-GluR2 AMPA-R LBD available for structural analysis. Standard cryoprotection protocols yielded high-resolution diffraction from flash-cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room-temperature data collection from capillary-mounted kainate cocrystals exhibited reproducible diffraction to better than 3 A resolution. Together, these crystals lay the foundation for a structural comparison of LBD-agonist interactions in distinct AMPA-R subunits

Domain Architecture of a Calcium-Permeable AMPA Receptor in a Ligand-Free Conformation

Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation

Small molecule conjugates with dimetal species for protein inhibition

Methods for targeting a protein by providing an inhibitor covalently linked to a rhodium(II) complex, introducing the inhibitor to the target protein and allowing the inhibitor and protein to interact. The rhodium(II) complex covalently linked to the inhibitor binds the target protein both inorganically and organically and forms stabilizing secondary contacts between the rhodium(II) complex and the protein

A photonic solution to exoplanet direct imaging via nulling interferometry

Direct imaging of exoplanets is vital for understanding star system formation and the evolutionary behaviour of exoplanets at large orbits. Typically, imaging a star system to find an exoplanet requires significant attenuation of the host star's high flux in order to detect the much weaker planetary light. The most common method to do this is coronagraphy, which blocks the starlight with an amplitude mask or a null inducing phase mask [1]. An alternative and attractive method is nulling interferometry where light from multiple telescopes are used to simultaneously form a high resolution image (or its Fourier components) and also to form a null in the vicinity of the host star, thereby attenuating it [2]. This has the advantage over coronagraphy that it is not limited to using a single telescope and is thus able to probe deeper into a star system by virtue of the higher resolution available by an interferometric array

Photonic mid-infrared nulling for exoplanet detection on a planar chalcogenide platform

The future of exoplanet detection lies in the mid-infrared (MIR). The MIR region contains the blackbody peak of both hot and habitable zone exoplanets, making the contrast between starlight and planet light less extreme. It is also the region where prominent chemical signatures indicative of life exist, such as ozone at 9.7 microns. At a wavelength of 4 microns the difference in emission between an Earth-like planet and a star like our own is 80 dB. However a jovian planet, at the same separation exhibits 60 dB of contrast, or only 20 dB if it is hot due to its formation energy or being close to its host star. A two dimensional nulling interferometer, made with chalcogenide glass, has been measured to produce a null of 20 dB, limited by scattered light. Measures to increase the null depth to the theoretical limit of 60 dB are discussed.Comment: Was published in SPIE: Optical and Infrared Interferometry and Imaging VI, Mike Ireland presente

Improved prediction equations for estimating height in adults from ethnically diverse backgrounds

Background & aims When body height cannot be measured, it can be predicted from ulna length (UL). However, commonly used published prediction equations may not provide useful estimates in adults from all ethnicities. This study aimed to evaluate the relationship between UL and height in adults from diverse ethnic groups and to consider whether this can be used to provide useful prediction equations for height in practice. Methods Standing height and UL were measured in 542 adults at seven UK locations. Ethnicity was self-defined using UK Census 2011 categories. Data were modelled to give two groups of height prediction equations based on UL, sex and ethnicity and these were tested against an independent dataset (n = 180). Results UL and height were significantly associated overall and in all groups except one with few participants (P = 0.059). The new equations yielded predicted height (H p) that was closer to measured height in the Asian and Black subgroups of the independent population than the Malnutrition Universal Screening Tool (MUST) equations. For Asian men, (H p (cm) = 3.26 UL (cm) + 83.58), mean difference from measured (95% confidence intervals) was −0.6 (−2.4, +1.2); Asian women, (H p = 3.26 UL + 77.62), mean difference +0.5 (−1.4, 2.4) cm. For Black men, H p = 3.14 UL + 85.80, −0.4 (−2.4, 1.7); Black women, H p = 3.14 UL + 79.55, −0.8 (−2.8, 1.2). These differences were not statistically significant while predictions from MUST equations were significantly different from measured height. Conclusions The new prediction equations provide an alternative for estimating height in adults from Asian and Black groups and give mean predicted values that are closer to measured height than MUST equations

Epoxide-Mediated CifR Repression of cif Gene Expression Utilizes Two Binding Sites in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees

Chalcogenide glass planar MIR couplers for future chip based Bracewell interferometers

Photonic integrated circuits are established as the technique of choice for a number of astronomical processing functions due to their compactness, high level of integration, low losses, and stability. Temperature control, mechanical vibration and acoustic noise become controllable for such a device enabling much more complex processing than can realistically be considered with bulk optics. To date the benefits have mainly been at wavelengths around 1550 nm but in the important Mid-Infrared region, standard photonic chips absorb light strongly. Chalcogenide glasses are well known for their transparency to beyond 10000 nm, and the first results from coupler devices intended for use in an interferometric nuller for exoplanetary observation in the Mid-Infrared L band (3800-4200 nm) are presented here showing that suitable performance can be obtained both theoretically and experimentally for the first fabricated devices operating at 4000 nm.Comment: in Proc. SPIE 9907, Optical and Infrared Interferometry and Imaging V, 990730 (August 4, 2016