Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions

We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid–ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.     

Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: Effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK_a values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of pK_a from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.     

The role of histidine residues in the alpha toxin of Clostridium perfringens

The α -toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Chemically-Synthesized Gene Encoding Modified Human Superoxpde Dismutase: Its Construction,Expression and Properties of the Product

The gene encoding modified human superoxide dismutax (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD.

Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl β-D-thiogalactoside by inserting the gene into the expression vector pKK223–3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codøn showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing.

The main isomer was purified to homogeneity and characterized. The h-SOD-Ala⁶ showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum. but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10°C. Thus. the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.     

Peripheral-zone treatments for plum curculio management: validation in commercial apple orchards

A field trial was conducted using an oil formulation of the entomopathogenic fungus, Metarhizium flavoviride Gams & Rozyspal (Deuteromycotina: Hyphomycetes), to control the variegated grasshopper, Zonocerus variegatus L. (Orthoptera: Pyrgomorphidae). The influence of dispersal and secondary pick-up from the spray residue on mortality was assessed by sampling insects from the treated plots at intervals and monitoring disease levels during subsequent incubation in the laboratory. The cumulative mortality curves showed the pathogen to be highly effective, even in samples taken 8 days after spraying. A model was developed to link the influence of mortality, incubation, secondary pick-up of spores and insect dispersal, on the shape of these cumulative mortality curves. The model proved useful for data received from an experiment using small plot sizes, where insect dispersal is a big problem. The spray residue had an important influence on overall insect mortality. The model also indicated a difference in speed of kill between field and laboratory samples, suggesting an incubation delay in the field.     

Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity

Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz-t targets the host ubiquitin-proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz-t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz-t, we generated double (K1,2R-AvrPiz-t), triple (K1,2,3R-AvrPiz-t) and lysine-free (LF-AvrPiz-t) mutants by mutating lysines into arginines in AvrPiz-t. LF-AvrPiz-t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co-expressed with APIP10 in Nicotiana benthamiana, LF-AvrPiz-t was more stable than AvrPiz-t and was less able to degrade APIP10. The avirulence of LF-AvrPiz-t on Piz-t:HA plants was less than that of AvrPiz-t, which led to resistance reduction and lower accumulation of the Piz-t:HA protein after inoculation with the LF-AvrPiz-t-carrying isolate. Chitin- and flg22-induced production of reactive oxygen species (ROS) was higher in LF-AvrPiz-t than in AvrPiz-t transgenic plants. In addition, LF-AvrPiz-t transgenic plants were less susceptible than AvrPiz-t transgenic plants to a virulent isolate. Furthermore, both AvrPiz-t and LF-AvrPiz-t interacted with OsRac1, but the suppression of OsRac1-mediated ROS generation by LF-AvrPiz-t was significantly lower than that by AvrPiz-t. Together, these results suggest that the lysine residues of AvrPiz-t are required for its avirulence and virulence functions in rice.     

Biomass,sugar, and bioethanol potential of sweet corn

Sweet corn is a widely distributed crop that generates agricultural waste without significant commercial value. In this study, we show that sweet corn varieties produce large amounts of residual biomass (10 t ha^?1) with high content of soluble sugars (25% of dry matter) in a short growing season (3 months). The potential ethanol production from structural and soluble sugars extracted from sweet corn stover reached up to 4400 l ha^?1 in the most productive hybrids, 33% of which (1500 l ha^?1) were obtained by direct fermentation of free sugars. We found wide genetic variation for biomass yield and soluble sugars content suggesting that those traits can be included as complementary traits in sweet corn breeding programs. Dual‐purpose sweet corn hybrids can have an added value for the farmers contributing to energy generation without affecting food supply or the environment.     

Sustainable limits to crop residue harvest for bioenergy: maintaining soil carbon in Australia's agricultural lands

The use of crop residues for bioenergy production needs to be carefully assessed because of the potential negative impact on the level of soil organic carbon (SOC) stocks. The impact varies with environmental conditions and crop management practices and needs to be considered when harvesting the residue for bioenergy productions. Here, we defined the sustainable harvest limits as the maximum rates that do not diminish SOC and quantified sustainable harvest limits for wheat residue across Australia's agricultural lands. We divided the study area into 9432 climate‐soil (CS) units and simulated the dynamics of SOC in a continuous wheat cropping system over 122 years (1889 – 2010) using the Agricultural Production Systems sIMulator (APSIM). We simulated management practices including six fertilization rates (0, 25, 50, 75, 100, and 200 kg N ha^?1) and five residue harvest rates (0, 25, 50, 75, and 100%). We mapped the sustainable limits for each fertilization rate and assessed the effects of fertilization and three key environmental variables – initial SOC, temperature, and precipitation – on sustainable residue harvest rates. We found that, with up to 75 kg N ha^?1 fertilization, up to 75% and 50% of crop residue could be sustainably harvested in south‐western and south‐eastern Australia, respectively. Higher fertilization rates achieved little further increase in sustainable residue harvest rates. Sustainable residue harvest rates were principally determined by climate and soil conditions, especially the initial SOC content and temperature. We conclude that environmental conditions and management practices should be considered to guide the harvest of crop residue for bioenergy production and thereby reduce greenhouse gas emissions during the life cycle of bioenergy production.     

Nitrogen and harvest effects on soil properties under rainfed switchgrass and no‐till corn over 9 years: implications for soil quality

Nitrogen fertilizer and harvest management will alter soils under bioenergy crop production and the long‐term effects of harvest timing and residue removal remain relatively unknown. Compared to no‐tilled corn (NT‐C, Zea mays L.), switchgrass (Panicum virgatum L.) is predicted to improve soil properties [i.e. soil organic C (SOC), soil microbial biomass (SMB‐C), and soil aggregation] due to its perennial nature and deep‐rooted growth form, but few explicit field comparisons exist. We assessed soil properties over 9 years for a rainfed study of N fertilizer rate (0, 60, 120, and 180 kg N ha^?1) and harvest management on switchgrass (harvested in August and postfrost) and NT‐C (with and without 50% stover removal) in eastern NE. We measured SOC, aggregate stability, SMB‐C, bulk density (BD), pH, P and K in the top 0–30 cm. Both NT‐C and switchgrass increased SMB‐C, SOC content, and aggregate stability over the 9 years, reflecting improvement from previous conventional management. However, the soils under switchgrass had double the percent aggregate stability, 1.3 times more microbial biomass, and a 5–8% decrease in bulk density in the 0–5 and 5–10 cm depths compared to NT‐C. After 9 years, cumulative decrease in available P was significantly greater beneath NT‐C (?24.0 kg P ha^?1) compared to switchgrass (?5.4 kg P ha^?1). When all measured soil parameters were included in the Soil Management Assessment Framework (SMAF), switchgrass improved soil quality index over time (ΔSQI) in all depths. NT‐C without residue removal did not affect ΔSQI, but 50% residue removal decreased ΔSQI (0–30 cm) due to reduced aggregate stability and SMB‐C. Even with best‐management practices such as NT, corn stover removal will have to be carefully managed to prevent soil degradation. Long‐term N and harvest management studies that include biological, chemical, and physical soil measurements are necessary to accurately assess bioenergy impacts on soils.     

Allosteric sites can be identified based on the residue–residue interaction energy difference

Allosteric drugs act at a distance to regulate protein functions. They have several advantages over conventional orthosteric drugs, including diverse regulation types and fewer side effects. However, the rational design of allosteric ligands remains a challenge, especially when it comes to the identification allosteric binding sites. As the binding of allosteric ligands may induce changes in the pattern of residue–residue interactions, we calculated the residue–residue interaction energies within the allosteric site based on the molecular mechanics generalized Born surface area energy decomposition scheme. Using a dataset of 17 allosteric proteins with structural data for both the apo and the ligand‐bound state available, we used conformational ensembles generated by molecular dynamics simulations to compute the differences in the residue–residue interaction energies in known allosteric sites from both states. For all the known sites, distinct interaction energy differences (>25%) were observed. We then used CAVITY, a binding site detection program to identify novel putative allosteric sites in the same proteins. This yielded a total of 31 “druggable binding sites,” of which 21 exhibited >25% difference in residue interaction energies, and were hence predicted as novel allosteric sites. Three of the predicted allosteric sites were supported by recent experimental studies. All the predicted sites may serve as novel allosteric sites for allosteric ligand design. Our study provides a computational method for identifying novel allosteric sites for allosteric drug design. Proteins 2015; 83:1375–1384. © 2014 Wiley Periodicals, Inc.