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  • Conference paper
    Girvan P, Teng X, Brooks NJ, Baldwin GS, Ying Let al., 2019,

    Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Implications

    , 63rd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 28A-28A, ISSN: 0006-3495
  • Journal article
    Rains JGD, ODonnelly K, Oliver T, Woscholski R, Long NJ, Barter LMCet al., 2019,

    Bicarbonate inhibition of carbonic anhydrase mimics hinders catalytic efficiency: Elucidating the mechanism and gaining insight toward improving speed and efficiency

    , ACS Catalysis, Vol: 9, Pages: 1353-1365, ISSN: 2155-5435

    Carbonic anhydrase (CA) mimics are often studied with a focus on the hydration of CO2 for atmospheric carbon capture. Consequently, the reverse reaction (dehydration of HCO3–) has received minimal attention, so much so that the rate-limiting step of the dehydration reaction in CA mimics is currently unknown. The rate-limiting step of the hydration reaction is reported to be the bicarbonate-bound intermediate step, and thus is susceptible to product inhibition. It is not, however, clear if this inhibition is a consequence of an increase in the rate of the competing dehydration reaction or resulting from the strong affinity of bicarbonate to the mimic. To address this, insight into the dehydration reaction kinetics is needed. We therefore report the most comprehensive study of a CA mimic to date. The dehydration profile of the fastest small-molecule CA mimic, ZnL1S, was characterized, and consequently evidence for the rate-limiting step for the dehydration reaction was seen to be the bicarbonate-bound intermediate step, much like the hydration reaction. This experimental validation of the rate-limiting step was achieved through a variety of methods including NMR experiments and the effect of inhibitors, substrate concentration, and metal center on activity. With this understanding, an improvement in the favorability of the rate-limiting step was achieved, resulting in decreased bicarbonate inhibition. Thus, an increase in the mimic’s kcat for both reactions was observed, resulting in the largest rate constants of any small-molecule CA mimic reported to date (28 093 and 579 M–1 s–1 for hydration and dehydration, respectively). Enzyme-like kcat/km values were obtained for ZnL1S (5.9 × 105 M–1 s–1 for CO2 hydration), and notably there is only a difference of 2.5 orders of magnitude from the enzyme, the closest of any CA mimic reported in the literature. The results from this work can be applied to the development and improvement

  • Journal article
    Cecchetti C, Pyle E, Byrne B, 2019,

    Transporter oligomerisation: roles in structure and function

    , Biochemical Society Transactions, Vol: 47, Pages: 433-440, ISSN: 0300-5127

    Oligomerisation is a key feature of integral membrane transporters with roles in structure, function and stability. In this review, we cover some very recent advances in our understanding of how oligomerisation affects these key transporter features, with emphasis on a few groups of transporters, including the nucleobase ascorbate transporters, neurotransmitter sodium symporters and major facilitator superfamily members.

  • Journal article
    Kaiser N, Mejuch T, Fedoryshchak R, Janning P, Tate EW, Waldmann Het al., 2019,

    Photoactivatable Myristic Acid Probes for UNC119-Cargo Interactions

    , CHEMBIOCHEM, Vol: 20, Pages: 134-139, ISSN: 1439-4227
  • Journal article
    Nielsen CD-T, Mooij WJ, Sale D, Rzepa HS, Bures J, Spivey ACet al., 2019,

    Reversibility and reactivity in an acid catalyzed cyclocondensation to give furanochromanes - a reaction at the "oxonium-Prins' vs. "ortho-quinone methide cycloaddition' mechanistic nexus

    , Chemical Science, Vol: 10, Pages: 406-412, ISSN: 2041-6520

    Herein we report a combined experimental and computational investigation of the acid catalyzed cyclocondensation reaction between styrenyl homoallylic alcohols and salicylaldehyde to form furanochromanes. We disclose a previously unreported isomerisation of the ‘unnatural’ trans-fused products to the diastereomeric ‘natural’ cis-fused congeners. Notwithstanding the appeal of assuming this corresponds to endo to exo isomerisation of Diels–Alder (D–A) adducts via concerted retro-cycloaddition/cycloaddition reactions of an in situ generated ortho-quinone methide with the styrenyl alkene, our combined Hammett/DFT study reveals a stepwise Prins-like process via discrete benzylic carbocation intermediates for all but the most electron deficient styrenes. As these reactions fortuitously lie at the intersection of these two mechanistic manifolds, it allows us to propose an experimentally determined indicative ρ+ value of ca. −3 as marking this nexus between a stepwise Prins-type pathway and a concerted cycloaddition reaction. This value should prove useful for categorising other reactions formally involving ‘ortho-quinomethides’, without the need for the extensive computation performed here. Logical optimisation of the reaction based upon the mechanistic insight led to the use of HFIP as an additive which enables exclusive formation of ‘natural’ cis-fused products with a ∼100-fold reaction rate increase and improved scope.

  • Journal article
    Goya Grocin A, Serwa R, Morales Sanfrutos J, Ritzefeld M, Tate Eet al., 2019,

    Whole proteome profiling of N-myristoyltransferase activity and inhibition using Sortase A

    , Molecular and Cellular Proteomics, Vol: 18, Pages: 115-126, ISSN: 1535-9476

    N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging.

  • Journal article
    Craven G, Affron D, Raymond P, Mann D, Armstrong Aet al., 2019,

    Vinyl sulfonamide synthesis for irreversible tethering via a novel α-selenoether protection strategy

    , MedChemComm, Vol: 10, Pages: 158-163, ISSN: 2040-2503

    Vinyl sulfonamides are valuable electrophiles for targeted protein modification and inhibition. We describe a novel approach to the synthesis of terminal vinyl sulfonamides which uses mild oxidative conditions to induce elimination of an α-selenoether masking group. The method complements traditional synthetic approaches and typically yields vinyl sulfonamides in high purity after aqueous work-up without requiring column chromatography of the final electrophilic product. The methodology is applied to the synthesis of covalent fragments for use in irreversible protein tethering and crucially enables the attachment of diverse fragments to the vinyl sulfonamide warhead via a chemical linker. Using thymidylate synthase as a model system, ethylene glycol is identified as an effective linker for irreversible protein tethering.

  • Journal article
    Menny A, Serna M, Boyd C, Gardner S, Joseph AP, Morgan BP, Topf M, Brooks NJ, Bubeck Det al., 2018,

    CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

    , Nature Communications, Vol: 9, ISSN: 2041-1723

    The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

  • Journal article
    Miller RM, Cabral J, Robles E, Brooks N, Ces Oet al., 2018,

    Crystallisation of sodium dodecyl sulfate–water micellar solutions with structurally similar additives: counterion variation

    , CrystEngComm, Vol: 20, Pages: 6834-6843, ISSN: 1466-8033

    The effects of a series of structurally similar sodium dodecyl sulfate (SDS) additives on the crystallisation of SDS–water micellar solutions were investigated using a combination of differential scanning calorimetry, dynamic light scattering, optical microscopy and inductively coupled plasma optical emission spectroscopy. Seven different counterions were chosen from groups 1 and 2 of the periodic table to replace the sodium on SDS: LDS, (SDS), KDS, RbDS, CsDS, Mg(DS)2, Ca(DS)2 and Sr(DS)2. Two representative temperature profileswere employed – linear cooling ramps at rate of 0.5 °C min−1 to determine near-equilibrium kinetics and transitions and isothermal holds at 6 °C to elucidate morphological changes. Crystallisation of the reference solution 20% SDS–H2O with 0.25, 1.0 and 2.5% additive was generally promoted or inhibited even at the lowest concentrations. Melting points however remained largely unchanged, suggesting that the additives predominantly had a kinetic rather than thermodynamic effect. ICP-OES measurements for the solutions containing 1% additive indicated that most of the additives were integrated into the SDS crystals which was reflected by morphological changes, including the formation of hexagonal and oval shaped crystals. Our results both quantify and provide a morphological insight into the effect of a series of additives on the crystallisation of micellar SDS solutions, which can readily form due to preferential Na exchange.

  • Journal article
    Barlow N, Kusumaatmaja H, Salehi-Reyhani A, Brooks N, Barter LMC, Flemming AJ, Ces Oet al., 2018,

    Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers

    , Journal of the Royal Society Interface, Vol: 15, ISSN: 1742-5662

    For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.

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