B21-High Throughput SAXS
B23-Circular Dichroism
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Philip
Bardelang
,
Ewan J.
Murray
,
Isobel
Blower
,
Sara
Zandomeneghi
,
Alice
Goode
,
Rohanah
Hussain
,
Divya
Kumari
,
Giuliano
Siligardi
,
Katsuaki
Inoue
,
Jeni
Luckett
,
James
Doutch
,
Jonas
Emsley
,
Weng C.
Chan
,
Philip
Hill
,
Paul
Williams
,
Boyan B.
Bonev
Diamond Proposal Number(s):
[5098, 12923, 13185, 13634, 15146]
Open Access
Abstract: Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.
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May 2023
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B21-High Throughput SAXS
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Ália
Dos Santos
,
Daniel E.
Rollins
,
Yukti
Hari-Gupta
,
Hannah
Mcarthur
,
Mingxue
Du
,
Sabrina Yong Zi
Ru
,
Kseniia
Pidlisna
,
Ane
Stranger
,
Faeeza
Lorgat
,
Danielle
Lambert
,
Ian
Brown
,
Kevin
Howland
,
Jesse
Aaron
,
Lin
Wang
,
Peter J. I.
Ellis
,
Teng-Leong
Chew
,
Marisa
Martin-Fernandez
,
Alice L. B.
Pyne
,
Christopher P.
Toseland
Diamond Proposal Number(s):
[16207]
Open Access
Abstract: NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear functions for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We find that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover nuclear roles for NDP52 in gene expression and DNA structure regulation.
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May 2023
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B21-High Throughput SAXS
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31323, 31668]
Open Access
Abstract: The allosteric regulation of biomolecules, such as enzymes, enables them to adapt and alter their conformation to fit specific substrates, expressing different functionalities in response to stimuli. Different stimuli can also trigger synthetic coordination cages to change their shape, size, and nuclearity by reconfiguring the dynamic metal–ligand bonds that hold them together. Here we demonstrate an abiological system consisting of different organic subcomponents and ZnII metal ions, which can respond to simple stimuli in complex ways. A ZnII20L12 dodecahedron transforms to give a larger ZnII30L12 icosidodecahedron through subcomponent exchange, as an aldehyde that forms bidentate ligands is displaced in favor of one that forms tridentate ligands together with a penta-amine subcomponent. In the presence of a chiral template guest, the same system that produced the icosidodecahedron instead gives a ZnII15L6 truncated rhombohedral architecture through enantioselective self-assembly. Under specific crystallization conditions, a guest induces a further reconfiguration of either the ZnII30L12 or ZnII15L6 cages to yield an unprecedented ZnII20L8 pseudo-truncated octahedral structure. The transformation network of these cages shows how large synthetic hosts can undergo structural adaptation through the application of chemical stimuli, opening pathways to broader applications.
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May 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26855]
Open Access
Abstract: Monkeypox virus (MPXV) is a double-stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non-endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost-effective antivirals could ease severe health outcomes. G-quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic-scale mapping of different MPXV isolates highlighted two conserved putative quadruplex-forming sequences MPXV-exclusive in 590 isolates. Subsequently, we assessed the G-quadruplex formation using circular dichroism spectroscopy and solution small-angle X-ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4-binding partners—Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small-molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G-quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G-quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.
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May 2023
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B21-High Throughput SAXS
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Biswanath
Hansda
,
Jhilam
Majumder
,
Biplab
Mondal
,
Akash
Chatterjee
,
Subhadeep
Das
,
Sourav
Kumar
,
Ratan
Gachhui
,
Valeria
Castelletto
,
Ian W.
Hamley
,
Prosenjit
Sen
,
Arindam
Banerjee
Diamond Proposal Number(s):
[29895]
Abstract: A histidine-based amphiphilic peptide (P) has been found to form an injectable transparent hydrogel in phosphate buffer solution over a pH range from 7.0 to 8.5 with an inherent antibacterial property. It also formed a hydrogel in water at pH = 6.7. The peptide self-assembles into a nanofibrillar network structure which is characterized by high-resolution transmission electron microscopy, field-emission scanning electron microscopy, atomic force microscopy, small-angle X-ray scattering, Fourier-transform infrared spectroscopy, and wide-angle powder X-ray diffraction. The hydrogel exhibits efficient antibacterial activity against both Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli). The minimum inhibitory concentration of the hydrogel ranges from 20 to 100 μg/mL. The hydrogel is capable of encapsulation of the drugs naproxen (a non-steroidal anti-inflammatory drug), amoxicillin (an antibiotic), and doxorubicin, (an anticancer drug), but, selectively and sustainably, the gel releases naproxen, 84% being released in 84 h and amoxicillin was released more or less in same manner with that of the naproxen. The hydrogel is biocompatible with HEK 293T cells as well as NIH (mouse fibroblast cell line) cells and thus has potential as a potent antibacterial and drug releasing agent. Another remarkable feature of this hydrogel is its magnification property like a convex lens.
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May 2023
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B21-High Throughput SAXS
I24-Microfocus Macromolecular Crystallography
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Kai
Wu
,
Tanya K.
Ronson
,
Pingru
Su
,
Zhi
Chen
,
Leonard
Goh
,
Andrew W.
Heard
,
Xiaopeng
Li
,
Fabian
Klautzsch
,
Christoph A.
Schalley
,
Mladen
Vinković
,
Jonathan R.
Nitschke
Diamond Proposal Number(s):
[31323, 31668]
Abstract: Biological encapsulants, such as viral capsids and ferritin protein cages, use many identical subunits to tile the surface of a polyhedron. Inspired by these natural systems, synthetic chemists have prepared artificial nanocages with well-defined shapes and cavities. Rational control over the self-assembly of discrete, nanometre-scale, hollow coordination cages composed of simple components remains challenging as a result of the entropic costs associated with binding many subunits together, difficulties in the error-correction processes associated with assembly and increasing surface energy as their size grows. Here we demonstrate the construction of nanocages of increasing size derived from a single pentatopic pyrrole-based subcomponent. Reasoned shifts in the preferred coordination number of the metal ions used, along with the denticity and steric hindrance of the ligands, enabled the generation of progressively larger cages. These structural changes of the cages are reminiscent of the differences in the folding of proteins caused by minor variations in their amino acid sequences; understanding how they affect capsule structure and thus cavity size may help to elucidate the construction principles for larger and functional capsules, capable of binding and carrying large biomolecules as cargoes.
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Apr 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[28806, 28287]
Abstract: Bioinspired de novo self-assembling peptides have been widely used for the development of soft biomaterials for a wide variety of biomedical and pharmaceutical applications, such as cell scaffolding for tissue engineering1, controlled and localised drug delivery2, biosensing3, and many others. The meticulous control of peptide-based nanomaterial properties over the length scale, by molecular design, remains the main challenge for tailoring biomaterials properties to meet the application needs. In our group, we have recently adopted a minimalistic molecular engineering approach for the development of Ultrashort Ionic-complementary Constrained Peptides (UICPs), which were rationally designed to self-assemble into amphiphilic β-sheet nanofibers with unique hydrogelation properties and surface activity.4 We have previously demonstrated the crucial role played by aromatic stacking for the formation and thermodynamic stabilisation of UICP β-sheet structures. Herein, we will show how charge interactions can be manipulated for fine tuning molecular self-assembly, morphology and size of nanofibrous structures formation and viscoelasticity of UICP hydrogels.
A library of 18 peptide sequences (4-5 residues long) was developed to study the effect of the sequence net charge, charge density distribution, reversal of charge order and ionic self-complementarity on their propensity towards self-assembly and gelation. Interestingly, 12 of these peptides self-assembled into β-sheet nanofibrous structures forming hydrogels at pH 4.5-5, as confirmed by ATR-FTIR, SEM, TEM, SAXS and oscillatory rheology. Full control over β-sheet content (ranging from ~30-80%), fibre morphology (thin fibrils, thick straight fibre bundles, twisted helical nanofibres, flat nanoribbons and nanotubes) and sizes (~4-67 nm in diameter), as well as gelation (critical gelation concentrations ranging from <7.5 to >100 mM) and viscoelastic properties (storage moduli G’ ~0.1-100 KPa) was achieved by the careful positioning of both Glu and Lys residues at both C- and N-termini, in the sequence core and on both the hydrophilic and hydrophobic faces of the peptide chain. In essence, this design approach enabled/disabled lateral growth along the β-sheet ladder via electrostatic attraction (counter charge, anion-pi and cation-pi)/repulsion, hence controlling fibre thickness, morphology, entanglement, and the resulting viscoelasticity of the system. Our UICPs platform thus provides the flexibility in peptide molecular design for the manufacturing of soft biomaterials with versatile properties that can be in future tailored to the relevant biomedical application.
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Apr 2023
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B21-High Throughput SAXS
DL-SAXS-Offline SAXS and Sample Environment Development
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Diamond Proposal Number(s):
[27756, 29720]
Open Access
Abstract: Atherosclerosis is often described as a single disease entity; however, the morphology of each plaque is unique to the individual. The field currently lacks a technique that can discriminate stable from unstable plaques, to identify those at risk of a thromboembolic event. Small- and wide-angle X-ray scattering (SAXS/WAXS) holds the potential to be able to identify key materials present in a plaque, such as cholesterol species, collagen, low-density lipoproteins (LDLs), and hydroxyapatite. Protocols have been established for the preparation of excised human atherosclerotic tissue that are investigated herein. This includes the fixing, sectioning, and substrate selection of the sample. Through several sample preparation methods, vast improvements have been made to sample-to-noise ratio and background subtraction.
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Apr 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Bornaviruses are RNA viruses with a mammalian, reptilian, and avian host range. The viruses infect neuronal cells and in rare cases cause a lethal encephalitis. The family Bornaviridae are part of the Mononegavirales order of viruses, which contain a nonsegmented viral genome. Mononegavirales encode a viral phosphoprotein (P) that binds both the viral polymerase (L) and the viral nucleoprotein (N). The P protein acts as a molecular chaperone and is required for the formation of a functional replication/transcription complex. In this study, the structure of the oligomerization domain of the phosphoprotein determined by X-ray crystallography is reported. The structural results are complemented with biophysical characterization using circular dichroism, differential scanning calorimetry and small-angle X-ray scattering. The data reveal the phosphoprotein to assemble into a stable tetramer, with the regions outside the oligomerization domain remaining highly flexible. A helix-breaking motif is observed between the α-helices at the midpoint of the oligomerization domain that appears to be conserved across the Bornaviridae. These data provide information on an important component of the bornavirus replication complex.
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Mar 2023
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B21-High Throughput SAXS
B23-Circular Dichroism
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Alessandro
Paciaroni
,
Valeria
Libera
,
Francesca
Ripanti
,
Andrea
Orecchini
,
Caterina
Petrillo
,
Daniela
Francisci
,
Elisabetta
Schiaroli
,
Samuele
Sabbatini
,
Anna
Gidari
,
Elisa
Bianconi
,
Antonio
Macchiarulo
,
Rohanah
Hussain
,
Lucia
Silvestrini
,
Paolo
Moretti
,
Norhan
Belhaj
,
Matteo
Vercelli
,
Yessica
Roque
,
Paolo
Mariani
,
Lucia
Comez
,
Francesco
Spinozzi
Diamond Proposal Number(s):
[29982, 32331]
Open Access
Abstract: The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1’ and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.
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Mar 2023
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