Role of a topologically conserved Isoleucine in the structure and function of Glutathione Transferases

Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of ScienceProteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding

The Post-Merger Magnetized Evolution of White Dwarf Binaries: The Double-Degenerate Channel of Sub-Chandrasekhar Type Ia Supernovae and the Formation of Magnetized White Dwarfs

Type Ia supernovae (SNe Ia) play a crucial role as standardizable cosmological candles, though the nature of their progenitors is a subject of active investigation. Recent observational and theoretical work has pointed to merging white dwarf binaries, referred to as the double-degenerate channel, as the possible progenitor systems for some SNe Ia. Additionally, recent theoretical work suggests that mergers which fail to detonate may produce magnetized, rapidly-rotating white dwarfs. In this paper, we present the first multidimensional simulations of the post-merger evolution of white dwarf binaries to include the effect of the magnetic field. In these systems, the two white dwarfs complete a final merger on a dynamical timescale, and are tidally disrupted, producing a rapidly-rotating white dwarf merger surrounded by a hot corona and a thick, differentially-rotating disk. The disk is strongly susceptible to the magnetorotational instability (MRI), and we demonstrate that this leads to the rapid growth of an initially dynamically weak magnetic field in the disk, the spin-down of the white dwarf merger, and to the subsequent central ignition of the white dwarf merger. Additionally, these magnetized models exhibit new features not present in prior hydrodynamic studies of white dwarf mergers, including the development of MRI turbulence in the hot disk, magnetized outflows carrying a significant fraction of the disk mass, and the magnetization of the white dwarf merger to field strengths $\sim 2 \times 10^8$ G. We discuss the impact of our findings on the origins, circumstellar media, and observed properties of SNe Ia and magnetized white dwarfs.Comment: Accepted ApJ version published on 8/20/13, with significant additional text added discussing the nature of the magnetized outflows, and possible CSM observational features relevant to NaID detection

Efficacy of Herbicides When Spray Solution Application Is Delayed

Information is limited concerning the impact of delaying applications of pesticides after solution preparation on efficacy. Experiments were conducted to determine weed control when diclosulam, dimethenamid-P, flumioxazin, fomesafen, imazethapyr, pendimethalin, and S-metolachlor were applied preemergence the day of solution preparation or 3, 6, and 9 days after solution preparation. Herbicide solutions were applied on the same day regardless of when prepared. Control of broadleaf signalgrass, common lambsquarters, entireleaf morningglory, and Palmer amaranth by these herbicides was not reduced regardless of when herbicide solutions were prepared. Surprisingly entireleaf morningglory control by all herbicides increased when herbicide application was delayed by 9 days. In separate experiments, control of broadleaf signalgrass by clethodim, common ragweed by glyphosate and lactofen, entireleaf morningglory by lactofen, Italian rye grass by glyphosate and paraquat, and Palmer amaranth by atrazine, dicamba, glufosinate, glyphosate, imazethapyr, lactofen, and 2,4-D was affected more by increase in weed size due to delayed application than the time between solution preparation and application

Flue-cured tobacco and Cl rates : implications on yield, quality, and nutrient concentration

The increase in flue-cured tobacco (Nicotiana tabacumL.) yields in recent decadesdue to genetic improvements of new cultivars and management technologies mayincrease the plant demand for Cl, and the increased dry mass may dilute Cl concentra-tion, thereby reducing negative effects. This study evaluated the effect of increasingdoses of Cl on tobacco production, quality, and chemical composition of leaves, infour growing environments located at research stations where flue-cured tobacco isproduced in North Carolina. The treatments consisted of 11 rates of Cl (0, 11, 22,34, 45, 56, 67, 78, 90, 101, and 112 kg ha−1) in each growing environment, with fourreplications in a randomized complete block design. The yield and visual quality,total alkaloids, and reducing sugars concentrations of cured leaf were determined.In addition, the concentration of selected nutrients (N, P, K, Ca, Mg, S, and Cl) andnitrate (NO3−) in tobacco leaves was measured in five different periods. Rates of Clup to 112 kg ha−1did not reduce the productivity or quality of flue-cured tobaccoin any environment. The Cl rate required to reach the threshold of 1.0% Cl contentin cured leaf was site-specific, being surpassed even in the control treatment at onelocation, or with Cl rates higher than 34 and 90 kg ha−1in two environments. Inone environment, the Cl rates increased tobacco yield, probably due the direct effectof Cl as a nutrient. Although the increasing Cl rates increased the reducing sugarsconcentration, visual quality was not attenuated

Spiral instability can drive thermonuclear explosions in binary white dwarf mergers

This is the final version of the article. Available from American Astronomical Society via the DOI in this record.Thermonuclear, or Type Ia supernovae (SNe Ia), originate from the explosion of carbon–oxygen white dwarfs, and serve as standardizable cosmological candles. However, despite their importance, the nature of the progenitor systems that give rise to SNe Ia has not been hitherto elucidated. Observational evidence favors the double-degenerate channel in which merging white dwarf binaries lead to SNe Ia. Furthermore, significant discrepancies exist between observations and theory, and to date, there has been no self-consistent merger model that yields a SNe Ia. Here we show that a spiral mode instability in the accretion disk formed during a binary white dwarf merger leads to a detonation on a dynamical timescale. This mechanism sheds light on how white dwarf mergers may frequently yield SNe Ia.We thank James Guillochon, Lars Bildsten, Matthew Wise, and Gunnar Martin Lellep for useful discussions and Matthias Aegenheyster for his contributions to the FLASH analysis codes. E.G.B. acknowledges support from MCINN grant AYA2011–23102, and from the European Union FEDER fund. The software used in this work was in part developed by the DOE NNSA-ASC OASCR Flash Center at the University of Chicago. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1053575. Simulations at UMass Dartmouth were performed on a computer cluster supported by NSF grant CNS-0959382 and AFOSR DURIP grant FA9550-10-1-0354. This research has made use of NASA's Astrophysics Data System and the yt astrophysics analysis software suite Turk et al. (2011). R.T.F. is grateful to have had the opportunity to complete this paper during a visit to the Kavli Institute for Theoretical Physics, which is supported in part by the National Science Foundation under grant No. NSF PHY11-25915

One-armed spiral instability in double-degenerate post-merger accretion disks

This is the author accepted manuscript. The final version is available from IOP Publishing via the DOI in this record.Increasing observational and theoretical evidence points to binary white dwarf mergers as the origin of some if not most normal Type Ia supernovae (SNe Ia). In this paper, we discuss the post-merger evolution of binary white dwarf (WD) mergers, and their relevance to the double-degenerate channel of SNe Ia. We present 3D simulations of carbon-oxygen (C/O) WD binary systems undergoing unstable mass transfer, varying both the total mass and the mass ratio. We demonstrate that these systems generally give rise to a one-armed gravitational spiral instability. The spiral density modes transport mass and angular momentum in the disk even in the absence of a magnetic field, and are most pronounced for secondary-to-primary mass ratios larger than 0.6. We further analyze carbon burning in these systems to assess the possibility of detonation. Unlike the case of a 1.1 + 1.0M C/O WD binary, we find that WD binary systems with lower mass and smaller mass ratios do not detonate as SNe Ia up to ∼ 8−22 outer dynamical times. Two additional models do however undergo net heating, and their secular increase in temperature could possibly result in a detonation on timescales longer than those considered hereWe thank James Guillochon, Daan Van Rossum, Chris Byrohl, and Pranav Dave for useful discussions. We also would like to thank the anonymous reviewer for their useful comments and insights. The work of EG-B, GA-S and PL-A was partially funded by MINECO AYA2014-59084-P grant and by the AGAUR. The software used in this work was in part developed by the DOE NNSA-ASC OASCR Flash Center at the University of Chicago. This work used the Extreme Science and Engineering discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1053575. Simulations at UMass Dartmouth were performed on a computer cluster supported by NSF grant CNS-0959382 and AFOSR DURIP grant FA9550-10-1-0354. RTF thanks the Institute for Theory and Computation at the Harvard-Smithsonian Center for Astrophysics, and the Kavli Institute for Theoretical Physics, supported in part by the national Science Foundation under grant NSF PHY11-25915, for visiting support during which this work was completed. This research has made use of resources from NASA’s Astrophysics Data System and the yt astrophysics analysis software suite (Turk et al. 2011)

2015-2016 Burley and Dark Tobacco Production Guide

Burley and dark tobacco growers in the U.S. make hundreds of decisions every growing season that impact the yield and quality of the crops that they produce. These decisions may include choosing appropriate varieties, planning effective pest control measures or perhaps deciding the best time to top or harvest a crop. Increasingly, tobacco growers are being required by the industry to record and justify their management decisions and actions. The most comprehensive example of this is the U.S. Tobacco Good Agricultural Practices (GAP) program that was initiated during the 2013 growing season and expanded in 2014. Under this program, all growers who sell tobacco to GAP Connections member organizations are required to attend training sessions on the principals of GAP and to keep detailed records of their production practices. Training requirements may change, but growers are currently required to attend training every season in which they plan to sell tobacco. Additional information about U.S. Tobacco GAP can be found by contacting GAP Connections. The written U.S. Tobacco GAP guidelines often refer growers to “University Tobacco Production Guides” for specific recommendations regarding management decisions. The information and recommendations provided in this guide have been developed and reviewed by tobacco production specialists and scientists at the University of Kentucky, University of Tennessee, Virginia Tech, and North Carolina State University. The purpose of this multi-state guide is to provide all burley and dark tobacco growers with the most current research-based recommendations for the production of high-yielding, high-quality tobacco. The guide provides advice on industry-accepted practices that may be applied across the burley and dark tobacco growing regions, although in some cases, growers may be referred to their local extension offices for additional information relevant to their specific situation