Dissecting the Functions of Conserved Prolines within Transmembrane Helices of the D2 Dopamine Receptor

G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing α-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis–trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P210^(5.50), a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures

Quasiperpendicular high Mach number Shocks

Shock waves exist throughout the universe and are fundamental to understanding the nature of collisionless plasmas. Reformation is a process, driven by microphysics, which typically occurs at high Mach number supercritical shocks. While ongoing studies have investigated this process extensively both theoretically and via simulations, their observations remain few and far between. In this letter we present a study of very high Mach number shocks in a parameter space that has been poorly explored and we identify reformation using in situ magnetic field observations from the Cassini spacecraft at 10 AU. This has given us an insight into quasi-perpendicular shocks across two orders of magnitude in Alfven Mach number (MA) which could potentially bridge the gap between modest terrestrial shocks and more exotic astrophysical shocks. For the first time, we show evidence for cyclic reformation controlled by specular ion reflection occurring at the predicted timescale of ~0.3 {\tau}c, where {\tau}c is the ion gyroperiod. In addition, we experimentally reveal the relationship between reformation and MA and focus on the magnetic structure of such shocks to further show that for the same MA, a reforming shock exhibits stronger magnetic field amplification than a shock that is not reforming.Comment: Accepted and Published in Physical Review Letters (2015

Charge-transfer heptamethine dyes for NIR singlet oxygen generation

A heptamethine-based charge-transfer dye was designed based on previous evidence of triplet state formation in orthogonal charge-transfer partners and calculations suggesting the formation of a charge-transfer state in heptamethine dye derivatives. An acridinium derivative of IR-1061 was subsequently synthesized and characterized, demonstrating photochemical reactivity at wavelengths over 1000 nm

Effects of Substituents on the Length of Central C(sp^3)-C(sp^3) Bond in Anthracene Photodimers and Related Molecules

Effects of substituents on the lengths of the central C–C single bond in the butterfly-shaped anthracene photodimers (1)–(7) and lepidopterenes (8) are studied. X-Ray analysis of the photodimer (10) of 9,10-difluoroanthracene gave a C(9)–C(10′) bond length of 1.631 (3)Å. An attempt to re-determine molecular structure of the photoisomer (5) of [2.2](9,10) anthracenophane (12) by neutron diffraction analysis is also reported [C(9)–C(10′): obs. 1.64(1), calc. 1.63(1)Å]. The D_2 structure that had been proposed for the minimum-energy conformation of (5) is questioned and the D_(2h) symmetric conformation is suggested on the basis of the diffraction results and MNDO calculations. The experimentally determined distances of the long central C–C bonds in these butterfly compounds including dianthronyl (9) are well reproduced by MNDO calculations with a standard deviation of 0.013 Å. Small but significant further elongation of the central C–C bond by up to 0.07 Å resulting from annulation of cyclobutane or cyclopentane ring in anthracene photodimers and from remote chlorine substitution in lepidopterene are interpreted in terms of the increased π→σ^* orbital interaction

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are vital to neuronal signaling, are implicated in important processes such as learning and memory, and are therapeutic targets for neural diseases. The α7 nAChR has been implicated in Alzheimer's disease and schizophrenia, and allosteric modulators have become one focus of drug development efforts. We investigate the mode of action of the α7-selective positive allosteric modulator, PNU-120596, and show that the higher potency of acetylcholine in the presence of PNU-120596 is not due to an altered agonist binding site. In addition, we propose several residues in the gating interface and transmembrane region that are functionally important to transduction of allosteric properties, and link PNU-120596, the acetylcholine binding region, and the receptor gate. These results suggest global protein stabilization from a communication network through several key residues that alter the gating equilibrium of the receptor while leaving the agonist binding properties unperturbed

High resolution radio observations of the colliding-wind binary WR140

Milli-arcsecond resolution Very Long Baseline Array (VLBA) observations of the archetype WR+O star colliding-wind binary (CWB) system WR140 are presented for 23 epochs between orbital phases 0.74 and 0.97. At 8.4 GHz, the emission in the wind-collision region (WCR) is clearly resolved as a bow-shaped arc that rotates as the orbit progresses. We interpret this rotation as due to the O star moving from SE to approximately E of the WR star, which leads to solutions for the orbit inclination of 122+/-5 deg, the longitude of the ascending node of 353+/-3 deg, and an orbit semi-major axis of 9.0+/-0.5 mas. The distance to WR140 is determined to be 1.85+/-0.16 kpc, which requires the O star to be a supergiant. The inclination implies the mass of the WR and O star to be 20+/-4 and 54+/-10 solar masses respectively. We determine a wind-momentum ratio of 0.22, with an expected half-opening angle for the WCR of 63 deg, consistent with 65+/-10 deg derived from the VLBA observations. Total flux measurements from Very Large Array (VLA) observations show the radio emission from WR140 is very closely the same from one orbit to the next, pointing strongly toward emission, absorption and cooling mechanism(s) that are controlled largely by the orbital motion. The synchrotron spectra evolve dramatically through the orbital phases observed, exhibiting both optically thin and optically thick emission. We discuss a number of absorption and cooling mechanisms that may determine the evolution of the synchrotron spectrum with orbital phase.Comment: Accepted by ApJ, to appear in v623, April 20, 2005. 14 pages, 13 figs, requires emulateapj.cls. A version with full resolution figs can be obtained from http://www.drao.nrc.ca/~smd/preprint/wr140_data.pd

Discovery of extended radio emission in the young cluster Wd1

We present 10 micron, ISO-SWS and Australia Telescope Compact Array observations of the region in the cluster Wd1 in Ara centred on the B[e] star Ara C. An ISO-SWS spectrum reveals emission from highly ionised species in the vicinity of the star, suggesting a secondary source of excitation in the region. We find strong radio emission at both 3.5cm and 6.3cm, with a total spatial extent of over 20 arcsec. The emission is found to be concentrated in two discrete structures, separated by 14''. The westerly source is resolved, with a spectral index indicative of thermal emission. The easterly source is clearly extended and nonthermal (synchrotron) in nature. Positionally, the B[e] star is found to coincide with the more compact radio source, while the southerly lobe of the extended source is coincident with Ara A, an M2 I star. Observation of the region at 10micron reveals strong emission with an almost identical spatial distribution to the radio emission. Ara C is found to have an extreme radio luminosity in comparison to prior radio observations of hot stars such as O and B supergiants and Wolf-Rayet stars, given the estimated distance to the cluster. An origin in a detatched shell of material around the central star is therefore suggested; however given the spatial extent of the emission, such a shell must be relatively young (10^3 yrs). The extended non thermal emission associated with the M star Ara A is unexpected; to the best of our knowledge this is a unique phenomenon. SAX (2-10keV) observations show no evidence of X-ray emission, which might be expected if a compact companion were present.Comment: 5 pages including encapsulated figures, figure 3 separate. Accepted for MNRAS pink page

A genome-integrated massively parallel reporter assay reveals DNA sequence determinants of cis-regulatory activity in neural cells

Recent large-scale genomics efforts to characterize the cis-regulatory sequences that orchestrate genome-wide expression patterns have produced impressive catalogues of putative regulatory elements. Most of these sequences have not been functionally tested, and our limited understanding of the non-coding genome prevents us from predicting which sequences are bona fide cis-regulatory elements. Recently, massively parallel reporter assays (MPRAs) have been deployed to measure the activity of putative cis-regulatory sequences in several biological contexts, each with specific advantages and distinct limitations. We developed LV-MPRA, a novel lentiviral-based, massively parallel reporter gene assay, to study the function of genome-integrated regulatory elements in any mammalian cell type; thus, making it possible to apply MPRAs in more biologically relevant contexts. We measured the activity of 2,600 sequences in U87 glioblastoma cells and human neural progenitor cells (hNPCs) and explored how regulatory activity is encoded in DNA sequence. We demonstrate that LV-MPRA can be applied to estimate the effects of local DNA sequence and regional chromatin on regulatory activity. Our data reveal that primary DNA sequence features, such as GC content and dinucleotide composition, accurately distinguish sequences with high activity from sequences with low activity in a full chromosomal context, and may also function in combination with different transcription factor binding sites to determine cell type specificity. We conclude that LV-MPRA will be an important tool for identifying cis-regulatory elements and stimulating new understanding about how the non-coding genome encodes information