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17 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	An engineered ubiquitin binding coiled coil peptide Pernille Vosbein , Paula Paredes Vergara , Danny T. Huang , Andrew Thomson Chemical Science 14 DOI: 10.1039/D4SC04204B Diamond Proposal Number(s): [28516] Open Access Show Abstract ▼ Abstract: Recognition of ubiquitin (Ub) is often mediated by small Ub binding domains such as the Ubiquitin Interacting Motif (UIM). Most Ub binding events are low affinity interactions, and designing stronger binders for Ub can be challenging. We here report the design of a short crosslinked coiled coil (CC) which is conformationally and chemically stable, and which can act as a scaffold to present the key binding residues from known UIM sequences. Doing so gives rise to a hybrid CC peptide that reconciles the important features of both UIM and CC sequences. We show by fluorescence polarization assays that this crosslinked ‘CC–UIM’ peptide exhibits enhanced binding to Ub compared to the original UIM sequence. Furthermore, we report a crystal structure of this peptide in complex with Ub. These studies show that preorganization of a small number of important binding residues onto a stable helical scaffold can be a successful strategy for binder design.	Sep 2024
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Constructing ion channels from water-soluble α-helical barrels Alistair J. Scott , Ai Niitsu , Huong T. Kratochvil , Eric J. M. Lang , Jason T. Sengel , William M. Dawson , Kozhinjampara R. Mahendran , Marco Mravic , Andrew Thomson , R. Leo Brady , Lijun Liu , Adrian J. Mulholland , Hagan Bayley , William F. Degrado , Mark I. Wallace , Derek N. Woolfson Nature Chemistry 537 DOI: 10.1038/s41557-021-00688-0 Show Abstract ▼ Abstract: The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide–lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.	May 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	How coiled-coil assemblies accommodate multiple aromatic residues Guto G. Rhys , William M. Dawson , Joseph L. Beesley , Freddie J. O. Martin , R. Leo Brady , Andrew Thomson , Derek N. Woolfson Biomacromolecules 2 DOI: 10.1021/acs.biomac.1c00131 Diamond Proposal Number(s): [12342] Show Abstract ▼ Abstract: Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely, α-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. Current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic α helices. The consequences of using aromatic residues—which would be useful for introducing structural probes, and binding and catalytic functionalities—into these interfaces are not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here, we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In another more complex structure, the residues are buried and form an extended hydrogen-bond network.	Apr 2021
I04-Macromolecular Crystallography	Navigating the structural landscape of de novo α-helical bundles Guto G. Rhys , Christopher W. Wood , Joseph L. Beesley , Nathan R. Zaccai , Antony Burton , R. Leo Brady , Andrew R. Thomson , Derek N. Woolfson Journal Of The American Chemical Society DOI: 10.1021/jacs.8b13354 Show Abstract ▼ Abstract: The association of amphipathic α helices in water leads to α-helical-bundle protein structures. However, the driving force for this—the hydrophobic effect—is not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increas-ingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundle—the α-helical coiled coils—relationships have been established that discriminate between all-parallel dimers, trimers and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less-well understood. Here, we describe a synthetic-peptide system that switches be-tween parallel hexamers and various up-down-up-down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high resolution with X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of α-helical coiled coils above the dimers and tri-mers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology.	May 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Maintaining and breaking symmetry in homomeric coiled-coil assemblies Guto G. Rhys , Christopher W. Wood , Eric J. M. Lang , Adrian J. Mulholland , R. Leo Brady , Andrew R. Thomson , Derek N. Woolfson Nature Communications 9 DOI: 10.1038/s41467-018-06391-y Diamond Proposal Number(s): [8922, 12342] Open Access Show Abstract ▼ Abstract: In coiled-coil (CC) protein structures α-helices wrap around one another to form rope-like assemblies. Most natural and designed CCs have two–four helices and cyclic (Cn) or dihedral (Dn) symmetry. Increasingly, CCs with five or more helices are being reported. A subset of these higher-order CCs is of interest as they have accessible central channels that can be functionalised; they are α-helical barrels. These extended cavities are surprising given the drive to maximise buried hydrophobic surfaces during protein folding and assembly in water. Here, we show that α-helical barrels can be maintained by the strategic placement of β-branched aliphatic residues lining the lumen. Otherwise, the structures collapse or adjust to give more-complex multi-helix assemblies without Cn or Dn symmetry. Nonetheless, the structural hallmark of CCs—namely, knobs-into-holes packing of side chains between helices—is maintained leading to classes of CCs hitherto unobserved in nature or accessed by design.	Oct 2018
I15-Extreme Conditions	The phase diagrams of KCaF3 and NaMgF3 by ab initio simulations Clément Jakymiw , Lidunka Vočadlo , David P. Dobson , Edward Bailey , Andrew R. Thomson , John P. Brodholt , Ian G. Wood , Alex Lindsay-Scott Physics And Chemistry Of Minerals 28 DOI: 10.1007/s00269-017-0920-3 Open Access Show Abstract ▼ Abstract: ABF3 compounds have been found to make valuable low-pressure analogues for high-pressure silicate phases that are present in the Earth’s deep interior and that may also occur in the interiors of exoplanets. The phase diagrams of two of these materials, KCaF3 and NaMgF3, have been investigated in detail by static ab initio computer simulations based on density functional theory. Six ABF3 polymorphs were considered, as follows: the orthorhombic perovskite structure (GdFeO3-type; space group Pbnm); the orthorhombic CaIrO3 structure (Cmcm; commonly referred to as the “post-perovskite” structure); the orthorhombic Sb2S3 and La2S3 structures (both Pmcn); the hexagonal structure previously suggested in computer simulations of NaMgF3 (P63/mmc); the monoclinic structure found to be intermediate between the perovskite and CaIrO3 structures in CaRhO3 (P21/m). Volumetric and axial equations of state of all phases considered are presented. For KCaF3, as expected, the perovskite phase is shown to be the most thermodynamically stable at atmospheric pressure. With increasing pressure, the relative stability of the KCaF3 phases then follows the sequence: perovskite → La2S3 structure → Sb2S3 structure → P63/mmc structure; the CaIrO3 structure is never the most stable form. Above about 2.6 GPa, however, none of the KCaF3 polymorphs are stable with respect to dissociation into KF and CaF2. The possibility that high-pressure KCaF3 polymorphs might exist metastably at 300 K, or might be stabilised by chemical substitution so as to occur within the standard operating range of a multi-anvil press, is briefly discussed. For NaMgF3, the transitions to the high-pressure phases occur at pressures outside the normal range of a multi-anvil press. Two different sequences of transitions had previously been suggested from computer simulations. With increasing pressure, we find that the relative stability of the NaMgF3 phases follows the sequence: perovskite → CaIrO3 structure → Sb2S3 structure → P63/mmc structure. However, only the perovskite and CaIrO3 structures are stable with respect to dissociation into NaF and MgF2.	Sep 2017
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Installing hydrolytic activity into a completely de novo protein framework Antony J. Burton , Andrew R. Thomson , William M. Dawson , R. Leo Brady , Derek N. Woolfson Nature Chemistry 8, 837 - 844 DOI: 10.1038/nchem.2555 Diamond Proposal Number(s): [8922] Show Abstract ▼ Abstract: The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine–histidine–glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.	Jul 2016
NONE-No attached Diamond beamline Mechanical Engineering	Preparations for the double-double bend achromat installation in Diamond Light Source Richard Walker , C Abraham , Cp Bailey , Riccardo Bartolini , P Coll , P Cox , Nigel Hammond , Mark Heron , Se Hughes , James Kay , I Martin , Shriram Mhaskar , A Miller , Aj Reed , Guenther Rehm , E Rial , Austen Rose , A Shahveh , Hugo Shiers , Andrew Thomson IPAC2016 DOI: 10.18429/JACoW-IPAC2016-WEPOW048 Open Access Show Abstract ▼ Abstract: We present the status of preparations for a major installation in the Diamond storage ring which is due to take place in 2016, namely the conversion of one cell of the ring from a double bend achromat (DBA) structure, to a double-DBA, or DDBA. We present results of measurements of the new narrow bore, high strength, quadrupoles and sextupoles, as well as the four new gradient dipoles. Fabrication of entirely new narrow-gap vacuum vessel strings, a mixture of copper and stainless steel is also described. The status of assembly of the two 7m long girders is presented, as well as other preparatory engineering, power supply, controls and high level software work.	Jun 2016
I15-Extreme Conditions	The stability of hydrous silicates in Earth's lower mantle: Experimental constraints from the systems MgO–SiO2–H2O and MgO–Al2O3–SiO2–H2O M. J. Walter , A. R. Thomson , W. Wang , O. T. Lord , J. Ross , S. C. Mcmahon , M. A. Baron , E. Melekhova , A. K. Kleppe , S. C. Kohn Chemical Geology 418, 16 – 29 DOI: 10.1016/j.chemgeo.2015.05.001 Diamond Proposal Number(s): [8669, 9788] Open Access Show Abstract ▼ Abstract: We performed laser-heated diamond anvil cell experiments on bulk compositions in the systems MgO–SiO2–H2O (MSH) and MgO–Al2O3–SiO2–H2O (MASH) that constrain the stability of hydrous phases in Earth's lower mantle. Phase identification by synchrotron powder diffraction reveals a consistent set of stability relations for the high-pressure, dense hydrous silicate phases D and H. In the MSH system phase D is stable to ~ 50 GPa, independent of temperature from ~ 1300 to 1700 K. Phase H becomes stable between 35 and 40 GPa, and the phase H out reaction occurs at ~ 55 GPa at 1600 K with a negative dT/dP slope of ~− 75 K/GPa. Between ~ 30 and 50 GPa dehydration melting occurs at ~ 1800 K with a flat dT/dP slope. A cusp along the solidus at ~ 50 GPa corresponds with the intersection of the subsolidus phase H out reaction, and the dT/dP melting slope steepens to ~ 15 K/GPa up to ~ 85 GPa.	Dec 2015
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Modular design of self-assembling peptide-based nanotubes Natasha Burgess , Thomas H. Sharp , Franziska Thomas , Christopher W. Wood , Andrew Thomson , Nathan Zaccai , Leo Brady , Louise Serpell , Derek N. Woolfson Journal Of The American Chemical Society 137 (33) DOI: 10.1021/jacs.5b03973 Diamond Proposal Number(s): [8922] Show Abstract ▼ Abstract: An ability to design peptide-based nanotubes (PNTs) rationally with defined and mutable internal channels would advance understanding of peptide self-assembly, and present new biomaterials for nanotechnology and medicine. PNTs have been made from Fmoc dipeptides, cyclic peptides, and lock-washer helical bundles. Here we show that blunt-ended α-helical barrels, that is, preassembled bundles of α-helices with central channels, can be used as building blocks for PNTs. This approach is general and systematic, and uses a set of de novo helical bundles as standards. One of these bundles, a hexameric α-helical barrel, assembles into highly ordered PNTs, for which we have determined a structure by combining cryo-transmission electron microscopy, X-ray fiber diffraction, and model building. The structure reveals that the overall symmetry of the peptide module plays a critical role in ripening and ordering of the supramolecular assembly. PNTs based on pentameric, hexameric, and heptameric α-helical barrels sequester hydrophobic dye within their lumens.	Aug 2015

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