The pH-dependent tertiary structure of a designed helix–loop–helix dimer

Background: De novo designed helix–loop–helix motifs can fold into well-defined tertiary structures if residues or groups of residues are incorporated at the helix–helix boundary to form helix-recognition sites that restrict the conformational degrees of freedom of the helical segments. Understanding the relationship between structure and function of conformational constraints therefore forms the basis for the engineering of non-natural proteins. This paper describes the design of an interhelical HisH+–Asp- hydrogen-bonded ion pair and the conformational stability of the folded helix–loop–helix motif.Results: GTD-C, a polypeptide with 43 amino acid residues, has been designed to fold into a hairpin helix–loop–helix motif that can dimerise to form a four-helix bundle. The folded motif is in slow conformational exchange on the NMR timescale and has a well-dispersed 1H NMR spectrum, a narrow temperature interval for thermal denaturation and a near-UV CD spectrum with some fine structure. The conformational stability is pH dependent with an optimum that corresponds to the pH for maximum formation of a hydrogen-bonded ion pair between HisH17+ in helix I and Asp27- in helix II.Conclusions: The formation of an interhelical salt bridge is strongly suggested by the pH dependence of a number of spectroscopic probes to generate a well-defined tertiary structure in a designed helix–loop–helix motif. The thermodynamic stability of the folded motif is not increased by the formation of the salt bridge, but neighbouring conformations are destabilised. The use of this novel design principle in combination with hydrophobic interactions that provide sufficient binding energy in the folded structure should be of general use in de novo design of native-like proteins

Mapping low-latitude stellar substructure with SEGUE photometry

Encircling the Milky Way at low latitudes, the Low Latitude Stream is a large stellar structure, the origin of which is as yet unknown. As part of the SEGUE survey, several photometric scans have been obtained that cross the Galactic plane, spread over a longitude range of 50 to 203 degrees. These data allow a systematic study of the structure of the Galaxy at low latitudes, where the Low Latitude Stream resides. We apply colour-magnitude diagram fitting techniques to map the stellar (sub)structure in these regions, enabling the detection of overdensities with respect to smooth models. These detections can be used to distinguish between different models of the Low Latitude Stream, and help to shed light on the nature of the system.Comment: To appear in the proceedings of IAU Symposium 254 "The Galaxy disk in a cosmological context", Copenhagen, June 200

The ducky^{2J} Mutation in Cacna2d2 Results in Reduced Spontaneous Purkinje Cell Activity and Altered Gene Expression

The mouse mutant ducky and its allele ducky^{2J} represent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the α₂δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α₂δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a 2 bp deletion in the coding region and a complete loss of α₂δ-2 protein. Here we show that du^{2J}/du^{2J} mice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C, du^{2J}/du^{2J} PCs show no spontaneous intrinsic activity. DU^{2J}/du^{2J} mice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du^{2J}/+ mice have a marked reduction in α₂δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du^{2J}/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α₂δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α₂δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma

Assessment and comparison of extreme sea levels and waves during the 2013/2014 storm season in two UK coastal regions

The extreme sea levels and waves experienced around the UK's coast during the 2013/2014 winter caused extensive coastal flooding and damage. In such circumstances, coastal managers seek to place such extremes in relation to the anticipated standards of flood protection, and the long-term recovery of the natural system. In this context, return periods are often used as a form of guidance. We therefore provide these levels for the winter storms, as well as discussing their application to the given data sets and case studies (two UK case study sites: Sefton, northwest England; and Suffolk, east England). We use tide gauge records and wave buoy data to compare the 2013/2014 storms with return periods from a national dataset, and also generate joint probabilities of sea level and waves, incorporating the recent events. The UK was hit at a national scale by the 2013/2014 storms, although the return periods differ with location. We also note that the 2013/2014 high water and waves were extreme due to the number of events, as well as the extremity of the 5 December 2013 "Xaver" storm, which had a very high return period at both case study sites. Our return period analysis shows that the national scale impact of this event is due to its coincidence with spring high tide at multiple locations as the tide and storm propagated across the continental shelf. Given that this event is such an outlier in the joint probability analyses of these observed data sets, and that the season saw several events in close succession, coastal defences appear to have provided a good level of protection. This type of assessment should be recorded alongside details of defence performance and upgrade, with other variables (e.g. river levels at estuarine locations) included and appropriate offsetting for linear trends (e.g. mean sea level rise) so that the storm-driven component of coastal flood events can be determined. Local offsetting of the mean trends in sea level allows long-term comparison of storm severity and also enables an assessment of how sea level rise is influencing return levels over time, which is important when considering long-term coastal resilience in strategic management plans

Assessment and comparison of extreme sea levels and waves during the 2013/14 storm season in two UK coastal regions

The extreme sea levels and waves experienced around the UK's coast during the 2013/14 winter caused extensive coastal flooding and damage. Coastal managers seek to place such extremes in relation to the anticipated standards of flood protection, and the long-term recovery of the natural system. In this context, return periods are often used as a form of guidance. This paper provides these levels for the winter storms, and discusses their application to the given data sets for two UK case study sites: Sefton, northwest England, and Suffolk, east England. Tide gauge records and wave buoy data were used to compare the 2013/14 storms with return periods from a national data set, and also joint probabilities of sea level and wave heights were generated, incorporating the recent events. The 2013/14 high waters and waves were extreme due to the number of events, as well as the extremity of the 5 December 2013 "Xaver" storm, which had a high return period at both case study sites. The national-scale impact of this event was due to its coincidence with spring high tide at multiple locations. Given that this event is such an outlier in the joint probability analyses of these observed data sets, and that the season saw several events in close succession, coastal defences appear to have provided a good level of protection. This type of assessment could in the future be recorded alongside defence performance and upgrade. Ideally other variables (e.g. river levels at estuarine locations) would also be included, and with appropriate offsetting for local trends (e.g. mean sea-level rise) so that the storm-driven component of coastal flood events can be determined. This could allow long-term comparison of storm severity, and an assessment of how sea-level rise influences return levels over time, which is important for consideration of coastal resilience in strategic management plans

Sediment dynamics of a nearshore sandbank: Results from TELEMAC-2D, TOMAWAC and SISYPHE modelling

Water Qualit

Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones <it>via </it>recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, <it>oriV</it>, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate <it>trans</it>-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer <it>oriV</it>-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a <it>P</it>_BAD-driven <it>trfA </it>gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.</p> <p>Results</p> <p>The <it>P</it>_BAD-driven copy of <it>cre </it>in EL350 was replaced seamlessly with a copy of <it>trfA</it>, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based <it>Caenorhabditis elegans </it>genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation.</p> <p>Conclusions</p> <p>By retrofitting EL350, an established 'recombineering' <it>E. coli </it>strain, with a tightly regulated copy of <it>trfA </it>we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of <it>oriV</it>-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.</p

The Star-forming Region NGC 346 in the Small Magellanic Cloud with Hubble Space Telescope ACS Observations. II. Photometric Study of the Intermediate-Age Star Cluster BS 90

We present the results of our investigation of the intermediate-age star cluster BS 90, located in the vicinity of the HII region N 66 in the SMC, observed with HST/ACS. The high-resolution data provide a unique opportunity for a very detailed photometric study performed on one of the rare intermediate-age rich SMC clusters. The complete set of observations is centered on the association NGC 346 and contains almost 100,000 stars down to V ~28 mag. In this study we focus on the northern part of the region, which covers almost the whole stellar content of BS 90. We construct its stellar surface density profile and derive structural parameters. Isochrone fits on the CMD of the cluster results in an age of about 4.5 Gyr. The luminosity function is constructed and the present-day mass function of BS 90 has been obtained using the mass-luminosity relation, derived from the isochrone models. We found a slope between -1.30 and -0.95, comparable or somewhat shallower than a typical Salpeter IMF. Examination of the radial dependence of the mass function shows a steeper slope at larger radial distances, indicating mass segregation in the cluster. The derived half-mass relaxation time of 0.95 Gyr suggests that the cluster is mass segregated due to its dynamical evolution. From the isochrone model fits we derive a metallicity for BS 90 of [Fe/H]=-0.72, which adds an important point to the age-metallicity relation of the SMC. We discuss our findings on this relation in comparison to other SMC clusters.Comment: Accepted for Publication in ApJ, 12 pages emulateapj TeX style, 10 figure

Observations of Stellar Objects at a Shell Boundary in the Star-Forming Complex in the Galaxy IC1613

The single region of ongoing star formation in the galaxy IC 1613 has been observed in order to reveal the nature of compact emission-line objects at the edges of two shells in the complex, identified earlier in H-alpha line images. The continuum images show these compact objects to be stars. Detailed spectroscopic observations of these stars and the surrounding nebulae were carried out with an integral field spectrograph MPFS mounted on the 6m telescope of the Special Astrophysical Observatory. The resulting stellar spectra were used to determine the spectral types and luminosity classes of the objects. An Of star we identified is the only object of this spectral type in IC 1613. The results of optical observations of the multi-shell complex are compared to 21cm radio observations. The shells harboring the stars at their boundaries constitute the most active part of the star-forming region. There is evidence that shocks have played an important role in the formation of the shells.Comment: 10 pages, 5 PS and 1 color JPEG figur