Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering

CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57. In order to probe the structural changes associated with activation, an approach which combines 19F NMR, protein engineering, and the known crystal structure of one conformer has been utilized. This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conformation of CheY. The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity. One of these 19F probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural "switch" in activation. 19F NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phsopho-CheY during NMR acquisition. Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule. However, phosphorylation did not perturb the probe of Lys109. The observed phosphorylated conformer is proposed to be the first step in the activation of CheY; later steps appear to perturb Lys109, as evidenced in the following paper. Together these results may give insight into the activation of other prokaryotic response regulators

The Frequency of Rapid Rotation Among K Giant Stars

We present the results of a search for unusually rapidly rotating giant stars in a large sample of K giants (~1300 stars) that had been spectroscopically monitored as potential targets for the Space Interferometry Mission's Astrometric Grid. The stars in this catalog are much fainter and typically more metal-poor than those of other catalogs of red giant star rotational velocities, but the spectra generally only have signal-to-noise (S/N) of ~20-60, making the measurement of the widths of individual lines difficult. To compensate for this, we have developed a cross-correlation method to derive rotational velocities in moderate S/N echelle spectra to efficiently probe this sample for rapid rotator candidates. We have discovered 28 new red giant rapid rotators as well as one extreme rapid rotator with a vsini of 86.4 km/s. Rapid rotators comprise 2.2% of our sample, which is consistent with other surveys of brighter, more metal-rich K giant stars. Although we find that the temperature distribution of rapid rotators is similar to that of the slow rotators, this may not be the case with the distributions of surface gravity and metallicity. The rapid rotators show a slight overabundance of low gravity stars and as a group are significantly more metal-poor than the slow rotators, which may indicate that the rotators are tidally-locked binaries.Comment: Accepted for publication in ApJ. 25 pages, 9 figures, 3 tables. Tables 1 and 2 are provided in their full form as plain text ancillary file

Extrasolar Planet Transits Observed at Kitt Peak National Observatory

We obtained J-, H- and JH-band photometry of known extrasolar planet transiting systems at the 2.1-m Kitt Peak National Observatory Telescope using the FLAMINGOS infrared camera between October 2008 and October 2011. From the derived lightcurves we have extracted the mid-transit times, transit depths and transit durations for these events. The precise mid-transit times obtained help improve the orbital periods and also constrain transit-time variations of the systems. For most cases the published system parameters successfully accounted for our observed lightcurves, but in some instances we derive improved planetary radii and orbital periods. We complemented our 2.1-m infrared observations using CCD z'-band and B-band photometry (plus two Hydrogen Alpha filter observations) obtained with the Kitt Peak Visitor's Center telescope, and with four H-band transits observed in October 2007 with the NSO's 1.6-m McMath-Pierce Solar Telescope. The principal highlights of our results are: 1) our ensemble of J-band planetary radii agree with optical radii, with the best-fit relation being: (Rp/R*)J = 0.0017 + 0.979 (Rp/R*)optical, 2) We observe star spot crossings during the transit of WASP-11/HAT-P-10, 3) we detect star spot crossings by HAT-P-11b (Kepler-3b), thus confirming that the magnetic evolution of the stellar active regions can be monitored even after the Kepler mission has ended, and 4) we confirm a grazing transit for HAT-P-27/WASP-40. In total we present 57 individual transits of 32 known exoplanet systems.Comment: 33 pages, 6 figures, accepted in Publications of the Astronomical Society of the Pacifi

Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering

The Escherichia coli CheY protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection of mutant CheY proteins was analyzed by behavioral assays, in vitro phosphorylation, and 19F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp13 in the CheY activation site results in activation, even for mutants which cannot be phosphorylated. Thus phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational "switch" in the activation site, is required for activation of CheY by either phosphorylation or mutation. The 19F NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081-13088) was again used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys109. Mutations which activate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformational change. Two striking contrasts were observed between activation by mutation and phosphorylation. (i) Each activating mutation generates a relatively localized perturbation in the activation site region, whereas phosphorylation triggers a global structural change. (ii) The perturbation of the Lys109 region observed for activating mutations is not detected in the phosphorylated protein. These results are consistent with a two-step model of activated CheY docking to the flagellar switch

Exploring impulsive solar magnetic energy release and particle acceleration with focused hard X-ray imaging spectroscopy

How impulsive magnetic energy release leads to solar eruptions and how those eruptions are energized and evolve are vital unsolved problems in Heliophysics. The standard model for solar eruptions summarizes our current understanding of these events. Magnetic energy in the corona is released through drastic restructuring of the magnetic field via reconnection. Electrons and ions are then accelerated by poorly understood processes. Theories include contracting loops, merging magnetic islands, stochastic acceleration, and turbulence at shocks, among others. Although this basic model is well established, the fundamental physics is poorly understood. HXR observations using grazing-incidence focusing optics can now probe all of the key regions of the standard model. These include two above-the-looptop (ALT) sources which bookend the reconnection region and are likely the sites of particle acceleration and direct heating. The science achievable by a direct HXR imaging instrument can be summarized by the following science questions and objectives which are some of the most outstanding issues in solar physics (1) How are particles accelerated at the Sun? (1a) Where are electrons accelerated and on what time scales? (1b) What fraction of electrons is accelerated out of the ambient medium? (2) How does magnetic energy release on the Sun lead to flares and eruptions? A Focusing Optics X-ray Solar Imager (FOXSI) instrument, which can be built now using proven technology and at modest cost, would enable revolutionary advancements in our understanding of impulsive magnetic energy release and particle acceleration, a process which is known to occur at the Sun but also throughout the Universe

The Role of Planet Accretion in Creating the Next Generation of Red Giant Rapid Rotators

Rapid rotation in field red giant stars is a relatively rare but well-studied phenomenon; here we investigate the potential role of planet accretion in spinning up these stars. Using Zahn's theory of tidal friction and stellar evolution models, we compute the decay of a planet's orbit into its evolving host star and the resulting transfer of angular momentum into the stellar convective envelope. This experiment assesses the frequency of planet ingestion and rapid rotation on the red giant branch (RGB) for a sample of 99 known exoplanet host stars. We find that the known exoplanets are indeed capable of creating rapid rotators; however, the expected fraction due to planet ingestion is only ~10% of the total seen in surveys of present-day red giants. Of the planets ingested, we find that those with smaller initial semimajor axes are more likely to create rapid rotators because these planets are accreted when the stellar moment of inertia is smallest. We also find that many planets may be ingested prior to the RGB phase, contrary to the expectation that accretion would generally occur when the stellar radii expand significantly as giants. Finally, our models suggest that the rapid rotation signal from ingested planets is most likely to be seen on the lower RGB, which is also where alternative mechanisms for spin-up, e.g., angular momentum dredged up from the stellar core, do not operate. Thus, rapid rotators on the lower RGB are the best candidates to search for definitive evidence of systems that have experienced planet accretion.Comment: 12 pages, 12 figures, accepted for publication in The Astrophysical Journa

Observable Signatures of Planet Accretion in Red Giant Stars I: Rapid Rotation and Light Element Replenishment

The orbital angular momentum of a close-orbiting giant planet can be sufficiently large that, if transferred to the envelope of the host star during the red giant branch (RGB) evolution, it can spin-up the star's rotation to unusually large speeds. This spin-up mechanism is one possible explanation for the rapid rotators detected among the population of generally slow-rotating red giant stars. These rapid rotators thus comprise a unique stellar sample suitable for searching for signatures of planet accretion in the form of unusual stellar abundances due to the dissemination of the accreted planet in the stellar envelope. In this study, we look for signatures of replenishment in the Li abundances and (to a lesser extent) 12C/13C, which are both normally lowered during RGB evolution. Accurate abundances were measured from high signal-to-noise echelle spectra for samples of both slow and rapid rotator red giant stars. We find that the rapid rotators are on average enriched in lithium compared to the slow rotators, but both groups of stars have identical distributions of 12C/13C within our measurement precision. Both of these abundance results are consistent with the accretion of planets of only a few Jupiter masses. We also explore alternative scenarios for understanding the most Li-rich stars in our sample---particularly Li regeneration during various stages of stellar evolution. Finally, we find that our stellar samples show non-standard abundances even at early RGB stages, suggesting that initial protostellar Li abundances and 12C/13C may be more variable than originally thought.Comment: Accepted for publication in the Astrophysical Journal. 29 pages in emulateapj format, including 16 figures and 12 tables. Tables 4 and 8 are provided in their entirety as plain text ancillary files (and will also be available in the electronic edition of ApJ

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls—fucosylated and sialylated human lactoferrin glycopeptides—and negative controls—high mannose glycopeptides from Saccharomyces cerevisiae—that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals

Soil Respiration in a Northeastern US Temperate Forest: A 22-Year Synthesis

To better understand how forest management, phenology, vegetation type, and actual and simulated climatic change affect seasonal and inter-annual variations in soil respiration (R$_{s}$), we analyzed more than 100,000 individual measurements of soil respiration from 23 studies conducted over 22 years at the Harvard Forest in Petersham, Massachusetts, USA. We also used 24 site-years of eddy-covariance measurements from two Harvard Forest sites to examine the relationship between soil and ecosystem respiration (R$_{e}$). R$_{s}$ was highly variable at all spatial (respiration collar to forest stand) and temporal (minutes to years) scales of measurement. The response of R$_{s}$ to experimental manipulations mimicking aspects of global change or aimed at partitioning R$_{s}$ into component fluxes ranged from −70% to +52%. The response appears to arise from variations in substrate availability induced by changes in the size of soil C pools and of belowground C fluxes or in environmental conditions. In some cases (e.g., logging, warming), the effect of experimental manipulations on R$_{s}$ was transient, but in other cases the time series were not long enough to rule out long-term changes in respiration rates. Inter-annual variations in weather and phenology induced variation among annual R$_{s}$ estimates of a magnitude similar to that of other drivers of global change (i.e., invasive insects, forest management practices, N deposition). At both eddy-covariance sites, aboveground respiration dominated R$_{e}$ early in the growing season, whereas belowground respiration dominated later. Unusual aboveground respiration patterns—high apparent rates of respiration during winter and very low rates in mid-to-late summer—at the Environmental Measurement Site suggest either bias in R$_{s}$ and R$_{e}$ estimates caused by differences in the spatial scale of processes influencing fluxes, or that additional research on the hard-to-measure fluxes (e.g., wintertime R$_{s}$, unaccounted losses of CO$_{2}$ from eddy covariance sites), daytime and nighttime canopy respiration and its impacts on estimates of R$_{e}$, and independent measurements of flux partitioning (e.g., aboveground plant respiration, isotopic partitioning) may yield insight into the unusually high and low fluxes. Overall, however, this data-rich analysis identifies important seasonal and experimental variations in R$_{s}$ and R$_{e}$ and in the partitioning of R$_{e}$ above- vs. belowground.Organismic and Evolutionary Biolog