B21-High Throughput SAXS
B23-Circular Dichroism
|
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.
|
May 2023
|
|
B21-High Throughput SAXS
B23-Circular Dichroism
|
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.
|
Mar 2023
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[29075, 31797]
Open Access
Abstract: Cyanine dyes are known to form H- and J-aggregates in aqueous solutions. Here we show that the cyanine dye, S0271, assembles in water into vortex induced chiral J-aggregates. The chirality of the J-aggregates depends on the directionality of the vortex. This study utilised both conventional benchtop CD spectropolarimeters and Mueller matrix polarimetry. It was found that J-aggregates have real chirality alongside linear dichroism and linear and circular birefringence. We identify the factors that are key to the formation of metastable chiral J-aggregates and propose a mechanism for their assembly.
|
Feb 2023
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[8475, 9567]
Open Access
Abstract: Inhaled nanoparticles (NPs) depositing in the alveolar region of the lung interact initially with a surfactant layer and in vitro studies have demonstrated that NPs can adversely affect the biophysical function of model pulmonary surfactants (PS), of which surfactant protein B (SP-B) is a key component. Other studies have demonstrated the potential for NPs to modify the structure and function of proteins. It was therefore hypothesised that NPs may affect the biophysical function of PS by modifying the structure of SP-B. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to explore the effect of various concentrations of gold nanoparticles (AuNPs) (5, 10, 20 nm), silver nanoparticles (AgNPs) (10 nm) and silver citrate on the secondary structure of surfactant protein B analogue, SP-B1–25, in a TFE/PB dispersion. For Au and Ag NPs the SRCD spectra indicated a concentration dependent reduction in the α-helical structure of SP-B1–25 (5 nm AuNP ≈ 10 nm AgNP ≫ 10 nm AuNP > 20 nm AuNP). For AuNPs the effect was greater for the 5 nm size, which was not fully explained by consideration of surface area. The impact of the 10 nm AgNPs was greater than that of the 10 nm AuNPs and the effect of AgNPs was greater than that of silver citrate at equivalent Ag mass concentrations. For 10 nm AuNPs, SRCD spectra for dispersions in, the more physiologically relevant, DPPC showed a similar concentration dependent pattern. The results demonstrate the potential for inhaled NPs to modify SP-B1–25 structure and thus potentially adversely impact the physiological function of the lung, however, further studies are necessary to confirm this.
|
Feb 2023
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[31392]
Open Access
Abstract: Designing the architecture of L-lysine-based polymeric structures is a highly challenging task that requires careful control of the amino acid reactive groups. Conventional processes to obtain branched polylysine need several steps and the addition of specific catalysts. In the present work, to gain a better understanding and control of the formation of L-lysine-based polymers, we have investigated the correlation between the protonation state of L-lysine and the corresponding hydrothermally grown structures. The samples have been characterized by combining optical spectroscopies, such as UV–Vis, fluorescence, and synchrotron radiation circular dichroism with structural analysis by Nuclear Magnetic Resonance, Fourier Transform Infrared spectroscopy, and dynamic light scattering. We have observed that aqueous precursor solutions with alkaline pHs promote the formation of branched structures. In contrast, high pHs favour the reactivity of the ε-amino groups leading to linear structures, as shown by circular dichroism analyses. On the other hand, acidic conditions trigger the branching of the amino acid. Interestingly, the polymeric forms of L-lysine emit in the blue because the increasing number of intermolecular hydrogen bonds promote the intermolecular charge transfer responsible for the emission. Understanding the correlation between the L-lysine charged states and the polymeric structures that could form controlling the protonation–deprotonation states of the amino acid opens the route to a refined design of polypeptide systems based on L-lysine.
|
Nov 2022
|
|
B23-Circular Dichroism
|
Karl
Frontzek
,
Marco
Bardelli
,
Assunta
Senatore
,
Anna
Henzi
,
Regina R.
Reimann
,
Seden
Bedir
,
Marika
Marino
,
Rohanah
Hussain
,
Simon
Jurt
,
Georg
Meisl
,
Mattia
Pedotti
,
Federico
Mazzola
,
Giuliano
Siligardi
,
Oliver
Zerbe
,
Marco
Losa
,
Tuomas
Knowles
,
Asvin
Lakkaraju
,
Caihong
Zhu
,
Petra
Schwarz
,
Simone
Hornemann
,
Matthew G.
Holt
,
Luca
Simonelli
,
Luca
Varani
,
Adriano
Aguzzi
Diamond Proposal Number(s):
[19680]
Open Access
Abstract: Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond (‘H-latch’), altering the flexibility of the α2–α3 and β2–α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity.
|
Aug 2022
|
|
B23-Circular Dichroism
I02-Macromolecular Crystallography
|
Open Access
Abstract: EfeUOB/M has been characterised in Pseudomonas syringae pathovar. syringae as a novel type of ferrous-iron transporter, consisting of an inner-membrane protein (EfeUPsy) and three periplasmic proteins (EfeOPsy, EfeMPsy and EfeBPsy). The role of an iron permease and peroxidase function has been identified for the EfeU and EfeB proteins, respectively, but the role of EfeO/M remains unclear. EfeMPsy is an ‘M75-only’ EfeO-like protein with a C-terminal peptidase-M75 domain (EfeOII/EfeM family). Herein, we report the 1.6 Å resolution crystal structure of EfeMPsy, the first structural report for an EfeM component of P. syringae pv. syringae. The structure possesses the bi-lobate architecture found in other bacterial periplasmic substrate/solute binding proteins. Metal binding studies, using SRCD and ICP-OES, reveal a preference of EfeMPsy for copper, iron and zinc. This work provides detailed knowledge of the structural scaffold, the metal site geometry, and the divalent metal binding potential of EfeM. This work provides crucial underpinning for a more detailed understanding of the role of EfeM/EfeO proteins and the peptidase-M75 domains in EfeUOB/M iron uptake systems in bacteria.
|
Mar 2022
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[23764, 25489]
Open Access
Abstract: The tau protein, a soluble protein associated with microtubules, which is involved in the assembly and stabilization of cytoskeletal elements, was found to form neurofibrillary tangles in different neurodegenerative diseases. Insoluble tau aggregates were observed to be organized in paired helical filaments (PHFs) and straight filaments (SFs). Recently, two small sequences (306–311 and 275–280) in the microtubule-binding region (MTBR), named PHF6 and PHF6*, respectively, were found to be essential for tau aggregation. Since a possible therapeutic approach consists of impairing amyloid formation either by stabilizing the native proteins or reducing the level of amyloid precursors, here we use synchrotron radiation circular dichroism (SRCD) at Diamond B23 beamline to evaluate the inhibitory effects of two small molecules, trehalose and ceftriaxone, against the aggregation of a small peptide containing the PHF6* sequence. Our results indicate that both these molecules, ceftriaxone and trehalose, increased the stability of the peptide toward aggregation, in particular that induced by heparin. With trehalose being present in many fruits, vegetables, algae and processed foods, these results support the need to investigate whether a diet richer in trehalose might exert a protective effect toward pathologies linked to protein misfolding.
|
Mar 2022
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[17621]
Open Access
Abstract: The study of the chiroptical properties of thin-film samples of two chiral benzo[1,2-b:4,5-b’]dithiophene-based oligothiophenes with optoelectronic properties was carried out at Diamond B23 beamline using the synchrotron radiation electronic circular dichroism imaging (SR-ECDi) facility. The experimental results support the hypothesis that the non-reciprocal circularly polarized absorption observed in these materials is due to the combined linear dichroism and linear birefringence contributions. The mapping at 50-µm spatial resolution revealed that several species coexist in the mesoscopic chiral domains likely due to different aggregated phases, which were identified in the films by SR-ECDi but could not be detected with benchtop ECD instruments
|
Mar 2022
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[17621, 19225]
Abstract: Solid-state ECD (ss-ECD) spectra of a model microcrystalline solid, finasteride, dispersed into a KCl pellet were recorded by using the synchrotron radiation source at the Diamond B23 beamline. Scanning a surface of 36 mm2 with a step of 0.5 mm, we measured a set of ECD imaging (ECDi) spectra very different from each other and from the ss-ECD recorded with a bench-top instrument (1 cm2 area). This is due to the anisotropic part of the ECD (ACD), which averages to zero in solution or on a large number of randomly oriented crystallites, but can otherwise be extremely large. Two-way singular value decomposition (SVD) analysis, through experimental and simulated TDDFT spectra, disclosed that the measured and theoretical principal components are in line with each other. This finding demonstrates that the observed isotropic ss-ECD spectrum is governed by the anisotropy of locally oriented crystals. It also introduces a new quality for ss-ECD measurements and opens a new future for probing and mapping chiral materials in the solid state such as active pharmaceutical ingredients (APIs).
|
Dec 2021
|
|
