• Controlled self-assembly of lambda-DNA networks with the synergistic effect of a DC electric field

      Gao, Mingyan; Hu, Jing; Wang, Ying; Liu, Mengnan; Wang, Jianfei; Song, Zhengxun; Xu, Hongmei; Hu, Cuihua; Wang, Zuobin; (American Chemical Society, 2019-11-04)
      Large-scale and morphologically controlled self-assembled λ-DNA networks were successfully constructed by the synergistic effect of a DC electric field. The effect of DNA concentration, direction, and intensity of the electric field, even the modification of the mica surface using Mg2+ on the characteristics of the as-prepared DNA networks, were investigated in detail by atomic force microscopy (AFM). It was found that the horizontal electric field was more advantageous to the formation of DNA networks with more regular structures. At the same concentration, the height of DNA network was not affected significantly by the intensity change of the horizontal electric field. The modification of Mg2+ on mica surface increased the aggregation of DNA molecules, which contributed to the morphological change of the DNA networks. Furthermore, DNA molecules were obviously stretched in both horizontal and vertical electric fields at low DNA concentrations.
    • Controlled self-assembly of λ-DNA networks with the synergistic effect of a DC electric field

      Gao, Mingyan; Hu, Jing; Wang, Ying; Liu, Mengnan; Wang, Jianfei; Song, Zhengxun; Xu, Hongmei; Hu, Cuihua; Wang, Zuobin; (American Chemical Society, 2019-11-04)
      Large-scale and morphologically controlled self-assembled λ-DNA networks were successfully constructed by the synergistic effect of a DC electric field. The effect of DNA concentration, direction, and intensity of the electric field, even the modification of the mica surface using Mg2+ on the characteristics of the as-prepared DNA networks, were investigated in detail by atomic force microscopy (AFM). It was found that the horizontal electric field was more advantageous to the formation of DNA networks with more regular structures. At the same concentration, the height of DNA network was not affected significantly by the intensity change of the horizontal electric field. The modification of Mg2+ on mica surface increased the aggregation of DNA molecules, which contributed to the morphological change of the DNA networks. Furthermore, DNA molecules were obviously stretched in both horizontal and vertical electric fields at low DNA concentrations.
    • Engineering nucleotide specificity of succinyl-CoA synthetase in blastocystis: the emerging role of gatekeeper residues

      Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M.; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh; Maytum, Robin; Pandey, Kailash C.; et al. (American Chemical Society, 2017-01-24)
      Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.
    • How helminth lipid-binding proteins offload their ligands to membranes: Differential mechanisms of fatty acid transfer by the ABA-1 polyprotein allergen and Ov-FAR-1 proteins of nematodes and Sj-FABPc of schistosomes

      McDermott, Lindsay C.; Kennedy, Malcolm W.; McManus, Donald P.; Bradley, Jannette E.; Cooper, Alan; Storch, Judith; Rutgers University; University of Glasgow; Queensland Institute for Medical Research; University of Nottingham (American Chemical Society, 2002-05-28)
      Three different classes of small lipid-binding protein (LBP) are found in helminth parasites. Although of similar size, the ABA-1A1 (also designated As-NPA-A1) and Ov-FAR-1 (formerly known as Ov20) proteins of nematodes are mainly alpha-helical and have no known structural counterparts in mammals, whereas Sj-FABPc of schistosomes is predicted to form a beta-barrel structure similar to the mammalian family of intracellular fatty acid binding proteins. The parasites that produce these proteins are unable to synthesize their own complex lipids and, instead, rely entirely upon their hosts for supply. As a first step in elucidating whether these helminth proteins are involved in the acquisition of host lipid, the process by which these LBPs deliver their ligands to acceptor membranes was examined, by comparing the rates and mechanisms of ligand transfer from the proteins to artificial phospholipid vesicles using a fluorescence resonance energy transfer assay. All three proteins bound the fluorescent fatty acid 2-(9-anthroyloxy)palmitic acid (2AP) similarly, but there were clear differences in the rates and mechanisms of fatty acid transfer. Sj-FABPc displayed a collisional mechanism; 2AP transfer rates increased with acceptor membrane concentration, were modulated by acceptor membrane charge, and were not diminished in the presence of increasing salt concentrations. In contrast, transfer of ligand from Ov-FAR-1 and ABA-1A1 involved an aqueous diffusion step; transfer rates from these proteins were not modulated by acceptor membrane concentration or charge but did decrease with the ionic strength of the buffer. Despite these differences, all of the proteins interacted directly with membranes, as determined using a cytochrome c competition assay, although Sj-FABPc interacted to a greater extent than did Ov-FAR-1 or rABA-1A1. Together, these results suggest that Sj-FABPc is most likely to be involved in the intracellular targeted transport and metabolism of fatty acids, whereas Ov-FAR-1 and ABA-1A1 may behave in a manner analogous to that of extracellular LBPs such as serum albumin and plasma retinol binding protein.
    • Imaging the substructures of individual IgE antibodies with atomic force microscopy

      Hu, Jing; Gao, Mingyan; Wang, Ying; Liu, Mengnan; Wang, Jianfei; Li, Jiani; Song, Zhengxun; Chen, Yujuan; Wang, Zuobin (American Chemical Society, 2019-10-29)
      The interaction between antibodies and substrates directly affects its conformation and thus its immune function. Therefore, it is desirable to study the structure of antibodies at the single molecule level. Herein, the substructures of Immunoglobulin E (IgE) on solid surfaces were investigated. For this purpose, the tapping-mode atomic force microscopy (AFM) was applied to observe the individual IgE substructures adsorbed onto Mg2+ and Na+ modified mica substrates in air. As expected, the AFM images revealed that the IgE antibodies exhibited different conformations on the surface of mica substrate, consisting of the four basic orientations: three domain, two equivalent domain, two unequal domain and single domain morphologies. Moreover, the differences of the different orientations in single IgE antibodies were also identified clearly.
    • Implementation of oxy-fuel combustion (OFC) technology in a gasoline direct injection (GDI) engine fueled with gasoline–ethanol blends

      Li, Xiang; Pei, Yiqiang; Li, Dayou; Ajmal, Tahmina; Aitouche, Abdel; Mobasheri, Raouf; Peng, Zhijun (American Chemical Society, 2021-10-27)
      Nowadays, to mitigate the global warming problem, the requirement of carbon neutrality has become more urgent. Oxy-fuel combustion (OFC) has been proposed as a promising way of carbon capture and storage (CCS) to eliminate carbon dioxide (CO2) emissions. This article explores the implementation of OFC technology in a practical gasoline direct injection (GDI) engine fueled with gasoline–ethanol blends, including E0 (gasoline), E25 (25% ethanol, 75% is gasoline in mass fraction), and E50 (50% ethanol, 50% is gasoline in mass fraction). The results show that with a fixed spark timing, φCA50 (where 50% fuel is burned), of E50 and E25 is about 4.5 and 1.9° later than that of E0, respectively. Ignition delay (θF) and combustion duration (θC) can be extended with the increase of the ethanol fraction in the blended fuel. With the increase of the oxygen mass fraction (OMF) from 23.3 to 29%, equivalent brake-specific fuel consumption (BSFCE) has a benefit of 2.12, 1.65, and 1.51% for E0, E25, and E50, respectively. The corresponding increase in brake-specific oxygen consumption (BSOC) is 21.83, 22.42, and 22.58%, respectively. Meanwhile, θF, θC, and the heat release rate (HRR) are not strongly affected by the OMF. With the increase of the OMF, the increment of θF is 0.7, 1.8, and 2.2° for E0, E25, and E50, respectively. θC is only extended by 1, 1.1, and 1.4°, respectively. Besides, by increasing the intake temperature (TI) from 298 to 358 K under all of the fuel conditions, BSFCE and BSOC present slight growth trends; θF and θC are slightly reduced; in the meantime, φCA50, φPmax (crank angle of peak cylinder pressure), and the position of the HRR peak are advanced by nearly 1°.
    • Isolation of an arsenate-respiring bacterium from a redox front in an arsenic-polluted aquifer in West Bengal, Bengal Basin

      Osborne, Thomas H.; McArthur, John H.; Sikdar, Pradip K.; Santini, Joanne M.; University College London; Indian Institute of Social Welfare and Business Management (American Chemical Society, 2015-03-03)
      Natural pollution of groundwater by arsenic adversely affects the health of tens of millions of people worldwide, with the deltaic aquifers of SE Asia being particularly polluted. The pollution is caused primarily by, or as a side reaction of, the microbial reduction of sedimentary Fe(III)-oxyhydroxides, but the organism(s) responsible for As release have not been isolated. Here we report the first isolation of a dissimilatory arsenate reducer from sediments of the Bengal Basin in West Bengal. The bacterium, here designated WB3, respires soluble arsenate and couples its reduction to the oxidation of acetate; WB3 is therefore implicated in the process of arsenic pollution of groundwater, which is largely by arsenite. The bacterium WB3 is also capable of reducing dissolved Fe(III) citrate, solid Fe(III)-oxyhydroxide, and elemental sulfur, using acetate as the electron donor. It is a member of the Desulfuromonas genus and possesses a dissimilatory arsenate reductase that was identified using degenerate polymerase chain reaction primers. The sediment from which WB3 was isolated was brown, Pleistocene sand at a depth of 35.2 m below ground level (mbgl). This level was some 3 cm below the boundary between the brown sands and overlying reduced, gray, Holocene aquifer sands. The color boundary is interpreted to be a reduction front that releases As for resorption downflow, yielding a high load of labile As sorbed to the sediment at a depth of 35.8 mbgl and concentrations of As in groundwater that reach >1000 μg/L.
    • Mutagenic and chemical modification of the ABA-1 allergen of the nematode Ascaris: consequences for structure and lipid binding properties

      McDermott, Lindsay C.; Moore, Joyce; Brass, Andrew; Price, Nicholas C.; Kelly, Sharon M.; Cooper, Alan; Kennedy, Malcolm W.; University of Glasgow; University of Manchester (American Chemical Society, 2001-08-21)
      The polyprotein allergens/antigens of nematodes (NPAs) are the only lipid binding proteins known to be produced as polyproteins. Cleavage of the large polyprotein precursors at regularly spaced proteinase cleavage sites produces 10 or 11 individual protein units of approximately 15 kDa. The sequences of these units are highly diverse within and between species, but there are five absolutely or strongly conserved amino acid positions (Trp15, Gln20, Leu42, Cys64, and Cys120). We have tested the role of these signature amino acids by mutational or chemical alteration of the ABA-1 protein of Ascaris, and examined the resulting modified proteins for perturbations of their lipid binding activities and structural integrity. Substitution of Trp15 and Gln20 both affect the stability of the protein in terms of resistance to thermal or chemical denaturation, but the ligand binding function is unaffected. Mutation of Leu42, however, disrupts both the protein's structural stability and functional integrity, as does chemical disruption of the disulfide bridge formed between Cys64 and Cys120. We also find that the C-terminal, but not the N-terminal, half of the protein binds fatty acids, indicating that the binding site may be confined to this part of the protein. This also supports the idea that the NPA units are themselves derived from an ancient duplication event, and that they may comprise two functionally distinct domains.
    • Tropomyosin isoforms show unexpected differential effects on actin polymerization

      Maytum, Robin; Dudekula, Khadar B.; University of Bedfordshire; University of Edinburgh (American Chemical Society, 2017-02-03)
      Tropomyosin is a rod-like coiled-coil protein that forms a continuous filament that is weakly associated, but firmly-attached to the surface of the actin filaments in all eukaryotic cells. Simple eukaryotes such as yeasts have only one or two different tropomyosin isoforms which are known to be essential and perform roles in regulating the actin cytoskeleton. However higher eukaryotes have larger numbers of tropomyosins, the number of which appear linked to organismal complexity. Mammals have 4 genes producing over 40 different isoforms by alternative splicing. In higher organisms tropomyosin is best known and characterized in the regulation of striated muscle contraction. The role of tropomyosin outside of muscle is less well understood. It is generally thought to have a regulatory role in controlling interactions of actin-binding proteins and in providing additional stability to actin-filaments. In the latter case has been considered that tropomyosin binds to actin-filaments some time after their formation, both making them mechanically stiffer and protecting them from breakdown. We have produced a range of recombinant tropomyosins from all four mammalian genes and characterized their actin-binding affinities in a cosedimentation assay. We have then used them to systematically study the effects of different isoforms of tropomyosin on actin polymerization for the first time. We have monitored actin polymerization by the well-characterised change in fluorescence of a pyrene-label attached to actin. Actin polymerisation is monitored by measuring the significant fluorescence enhancement on polymerization. Our results characterize the actin-affinities of some of the TPM3 and TPM4 isoforms for the first time, These are in the same general range as mammalian isoforms previously characterized by our group and others. We demonstrate differential effects of the different isoforms on actin-polymerisation for the first time. The data unexpectedly show the most significant effects of the different isoforms appears to be in the early initiation / elongation stages of polymerizations. This is unexpected as tropomyosin is only considered to have significant affinity for actin filaments through itself forming a polymer along the surface of an actin filament. Different isoforms appear capable of both enhancing and inhibiting the early stages of polymerization, with examples of the shorter 6-actin spanning TPM1 gene isoforms showing a significant reduction in the lag-phase of early polymerization. These differential effects on different isoforms provides a new role for tropomyosin in not only stabilizing filaments, but also in helping catalyze their formation.
    • Zn-alpha2-glycoprotein, an MHC class I-related glycoprotein regulator of adipose tissues: modification or abrogation of ligand binding by site-directed mutagenesis

      McDermott, Lindsay C.; Freel, June A.; West, Anthony P.; Bjorkman, Pamela J.; Kennedy, Malcolm W.; University of Glasgow; California Institute of Technology (American Chemical Society, 2006-01-31)
      Zn-alpha(2)-glycoprotein (ZAG) is a soluble lipid-mobilizing factor associated with cancer cachexia and is a novel adipokine. Its X-ray crystal structure reveals a poly(ethylene glycol) molecule, presumably substituting for a higher affinity natural ligand, occupying an apolar groove between its alpha(1) and alpha(2) domain helices that corresponds to the peptide binding groove in class I MHC proteins. We previously provided evidence that the groove is a binding site for hydrophobic ligands that may relate to the protein's signaling function and that the natural ligands are probably (polyunsaturated) fatty acid-like. Using fluorescence-based binding assays and site-directed mutagenesis, we now demonstrate formally that the groove is indeed the binding site for hydrophobic ligands. We also identify amino acid positions that are involved in ligand binding and those that control the shape and exposure to solvent of the binding site itself. Some of the mutants showed minimal effects on their binding potential, one showed enhanced binding, and several were completely nonbinding. Particularly notable is Arg-73, which projects into one end of the binding groove and is the sole charged amino acid adjacent to the ligand. Replacing this amino acid with alanine abolished ligand binding and closed the groove to solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic ligand. These data add weight to the distinctiveness of ZAG among MHC class I-like proteins in addition to providing defined binding-altered mutants for cellular signaling studies and potential medical applications.