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119 items found (0 items not approved), displaying 11 to 20.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
B23-Circular Dichroism	Enzyme–ligand interaction monitored by synchrotron radiation circular dichroism Rohanah Hussain , Charlotte S. Hughes , Giuliano Siligardi DOI: 10.1007/978-1-0716-0163-1_6 Diamond Proposal Number(s): [12182, 14484, 16778, 19680] Open Access Show Abstract ▼ Abstract: CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein–ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC. This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand. The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules to measure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.	Nov 2019
B23-Circular Dichroism	Giant photoinduced chirality in thin film Ge2Sb2Te5 Janaki Shanmugam , Konstantin B. Borisenko , Andrew Luers , Paul Ewart , Priyav Shah , Benjamin A. O. Williams , Christopher Craig , Daniel W. Hewak , Rohanah Hussain , Tamas Javorfi , Giuliano Siligardi , Michel Bosman , Angus I. Kirkland Physica Status Solidi (rrl) – Rapid Research Letters 1 DOI: 10.1002/pssr.201900449 Diamond Proposal Number(s): [9375, 15028] Show Abstract ▼ Abstract: Induction, tuning, or amplification of chirality in various classes of materials and probing their chiral response are subjects of growing research. Herein, a large chiral signal that is rapidly imprinted in achiral amorphous Ge2Sb2Te5 (GST) thin films measured using synchrotron circular dichroism spectroscopy is reported. The chirality is induced by illuminating the films with pulsed circularly polarized (chiral) laser light for less than 2 μs in total. The effects of laser fluence and film thickness on the chiral response are described. The correlation of the optical results with structural studies by electron diffraction and model simulations suggests that alignment of reamorphized fragments in the crystallized film along the electric field vector of the light forms the centers that are responsible for the observed chirality. These results suggest opportunities for practical applications of this phenomenon and provide avenues for further studies of chirality induction in materials with impact in a wide range of disciplines.	Oct 2019
B23-Circular Dichroism	Circular dichroism and synchrotron radiation circular dichroism applications to biomaterials Rohanah Hussain , Tamas Javorfi , Charlotte S. Hughes , Giuliano Siligardi DOI: 10.1007/978-3-030-28247-9_5 Diamond Proposal Number(s): [8714, 9786, 11563, 18011, 2023, 2074, 5718, 5950, 4980, 12182, 14484, 16778, 19680, 8681] Show Abstract ▼ Abstract: Circular dichroism is the differential absorption of left and right circularly polarized light (CPL) of chiral molecules. Molecules that do not superimpose their mirror images are chiral like L and D amino acids, of which the L enantiomers are the building blocks of proteins and analogously D-deoxyribose for DNA.	Sep 2019
B23-Circular Dichroism	Electronic circular dichroism imaging (CDi) maps local aggregation modes in thin films of chiral oligothiophenes Gianluigi Albano , Marcin Gorecki , Gennaro Pescitelli , Lorenzo Di Bari , Tamas Javorfi , Rohanah Hussain , Giuliano Siligardi New Journal Of Chemistry DOI: 10.1039/C9NJ02746G Diamond Proposal Number(s): [14986] Show Abstract ▼ Abstract: We highlight the power of electronic circular dichroism imaging (CDi), a technique developed at Diamond Light Source B23 beamline for synchrotron radiation circular dichroism (SRCD) to detect local domains of chiral supramolecular order in thin films of chiral oligomers and polymers. The highly brilliant and collimated beamlight of B23 enabled CDi at a spatial resolution of 0.1 mm, unattainable with benchtop electronic circular dichroism (ECD) spectropolarimeters. CDi is bridging the gap between standard ECD spectroscopy and conventional microscopy. Presently, we apply CDi to reveal the local polymorphism of chiral oligothiophene-based molecules. By extensive use of a post-acquisition data-analysis tool called similarity factor, we quantified the polymorphs revealing a manifold of aggregation pathways whose relative weight was function of the sample preparation protocol. This work uncovers the parameters that need to be optimised in order to obtain reproducible and controllable supramolecular structures in thin films, which is of paramount importance for materials with optoelectronic properties. Noteworthy, this analysis is based on the CDi measurement at high spatial resolution of the thin film of organic chiral semiconductor at the ultimate stage of preparation as the active layer of an optoelectronic device.	Aug 2019
B23-Circular Dichroism	The Tim-3-Galectin-9 pathway and its regulatory mechanisms in human breast cancer Inna M. Yasinska , Svetlana S. Sakhnevych , Ludmila Pavlova , Anette Teo Hansen Selnø , Ana Maria Teuscher Abeleira , Ouafa Benlaouer , Isabel Gonçalves Silva , Marianne Mosimann , Luca Varani , Marco Bardelli , Rohanah Hussain , Giuliano Siligardi , Dietmar Cholewa , Steffen M. Berger , Bernhard F. Gibbs , Yuri A. Ushkaryov , Elizaveta Fasler-kan , Elena Klenova , Vadim V. Sumbayev Frontiers In Immunology 10 DOI: 10.3389/fimmu.2019.01594 Diamond Proposal Number(s): [20755] Open Access Show Abstract ▼ Abstract: Human cancer cells operate a variety of effective molecular and signaling mechanisms which allow them to escape host immune surveillance and thus progress the disease. We have recently reported that the immune receptor Tim-3 and its natural ligand galectin-9 are involved in the immune escape of human acute myeloid leukemia (AML) cells. These cells use the neuronal receptor latrophilin 1 (LPHN1) and its ligand fibronectin leucine rich transmembrane protein 3 (FLRT3, and possibly other ligands) to trigger the pathway. We hypothesized that the Tim-3-galectin-9 pathway may be involved in the immune escape of cancer cells of different origins. We found that studied breast tumors expressed significantly higher levels of both galectin-9 and Tim-3 compared to healthy breast tissues of the same patients and that these proteins were co-localized. Increased levels of LPHN2 and expressions of LPHN3 as well as FLRT3 were also detected in breast tumor cells. Activation of this pathway facilitated the translocation of galectin-9 onto the tumor cell surface, however no secretion of galectin-9 by tumor cells was observed. Surface-based galectin-9 was able to protect breast carcinoma cells against cytotoxic T cell-induced death. Furthermore, we found that cell lines from brain, colorectal, kidney, blood/mast cell, liver, prostate, lung, and skin cancers expressed detectable amounts of both Tim-3 and galectin-9 proteins. The majority of cell lines expressed one of the LPHN isoforms and FLRT3. We conclude that the Tim-3-galectin-9 pathway is operated by a wide range of human cancer cells and is possibly involved in prevention of anti-tumor immunity.	Jul 2019
B23-Circular Dichroism	A temperature‐driven, reversible helical handedness inversion in peptaibol analogs tuned by the C‐terminal capping moiety Marta De Zotti , Victoria N. Syryamina , Rohanah Hussain , Edoardo Longo , Giuliano Siligardi , Sergei A. Dzuba , Lorenzo Stella , Fernando Formaggio Chembiochem DOI: 10.1002/cbic.201900235 Diamond Proposal Number(s): [13463, 11528, 14298] Show Abstract ▼ Abstract: Trichogin is a natural peptide endowed with antimicrobial and antitumor activity. A member of the peptaibol family, trichogin possesses a C‐terminal aminoalcohol. In the past, we substituted that moiety with a methyl ester for synthetic purposes and realized that this apparently slight modification causes great changes in the peptide bioactivity. Aiming at understanding the reasons behind such observations, we performed a detailed spectroscopic study on a number of trichogin analogs. In the present manuscript, we compare the data obtained from synchrotron radiation circular dichroism, NMR and fluorescence spectroscopy in organic solvents at cryogenic temperatures with those independently acquired by electron paramagnetic resonance spectroscopy at 80K. We reveal unambiguously the presence of a reversible, temperature‐driven screw‐sense interconversion from right‐handed to left‐handed helix that is determined by the C‐terminal capping moiety. Our data demonstrate for the first time the key role of a C‐terminal methyl ester in promoting peptide screw‐sense inversion.	May 2019
B23-Circular Dichroism	Selection of biophysical methods for characterisation of membrane proteins Tristan O. C. Kwan , Rosana Reis , Giuliano Siligardi , Rohanah Hussain , Harish Cheruvara , Isabel Moraes International Journal Of Molecular Sciences 20 DOI: 10.3390/ijms20102605 Diamond Proposal Number(s): [12182] Open Access Show Abstract ▼ Abstract: Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure–function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research.	May 2019
B23-Circular Dichroism	Targeting of basophil and mast cell pro-allergic reactivity using functionalised gold nanoparticles Inna M. Yasinska , Luigi Calzolai , Ulrike Raap , Rohanah Hussain , Giuliano Siligardi , Vadim V. Sumbayev , Bernhard F. Gibbs Frontiers In Pharmacology 10 DOI: 10.3389/fphar.2019.00333 Diamond Proposal Number(s): [12578] Open Access Show Abstract ▼ Abstract: Calcineurin inhibitors potentially prevent pro-allergic mediator release from basophils and mast cells but are rarely used systemically due to ubiquitous expressions of target signaling proteins. However, specific targeting of allergic effector cells with these inhibitors could circumvent unwanted side effects. We recently demonstrated the biocompatibility of gold nanoparticles (AuNPs) as a platform for non-toxic delivery of signaling inhibitors due to unique physicochemical properties of these nanomaterials. Since AuNPs can be conjugated with both anti-allergic drugs and antibodies or other proteins that specifically recognize basophils and mast cells, our aims were to assess specific targeting of allergic effector cell function using AuNPs conjugated with the calcineurin inhibitor ascomycin. Purified human basophils and LAD2 human mast cells were used for investigations with AuNPs conjugated either to CD203c antibodies or containing stem cell factor (SCF), respectively, which were amine-coupled to acidic groups of reduced glutathione (GSH). GSH was also used as a spacer for immobilization of ascomycin on the gold surface. AuNPs conjugated with anti-CD203c and ascomycin strikingly blocked IgE-dependent degranulation of both purified basophils and those present in mixed leukocyte preparations, suggesting specific targeting of these cells. In contrast, LAD2 mast cell responses were not inhibited using anti-CD203c-containing nanoconjugates but were when the conjugates contained SCF. Successful targeting of allergic effector cells using gold nanoconjugates indicates that this technology may have therapeutic potential for the treatment of allergies by specifically delivering highly effective signaling inhibitors with reduced side effects.	Mar 2019
B23-Circular Dichroism	Anisotropic circular dichroism of light-harvesting complex II in oriented lipid bilayers: Theory meets experiment Parveen Akhtar , Dominik Lindorfer , Monika Lingvay , Krzysztof Pawlak , Otto Zsiros , Giuliano Siligardi , Tamas Javorfi , Márta Dorogi , Bettina Ughy , Gyozo Garab , Thomas Renger , Petar H. Lambrev The Journal Of Physical Chemistry B DOI: 10.1021/acs.jpcb.8b12474 Diamond Proposal Number(s): [15094, 16232, 17698, 19120, 20644] Show Abstract ▼ Abstract: Anisotropic circular dichroism (ACD) spectroscopy of macroscopically aligned molecules reveals additional information about their excited states that is lost in the CD of randomly-oriented solutions. ACD spectra of light-harvesting complex II (LHCII)—the main peripheral antenna of photosystem II in plants—in oriented lipid bilayers, were recorded from the far-UV to the visible wavelength region. The ACD spectra show a drastically enhanced magnitude and level of detail compared to the isotropic CD spectra, resolving a greater number of bands and weak optical transitions. Exciton calculations show that the spectral features in the chlorophyll Qy region are well reproduced by an existing Hamiltonian for LHCII, providing further evidence for the identity of energy sinks at chlorophylls a603 and a610 in the stromal layer and chlorophylls a604 and a613 in the luminal layer. We propose ACD spectroscopy to be a valuable tool linking the three-dimensional structure and the photophysical properties of pigment–protein complexes.	Jan 2019
B23-Circular Dichroism	Characterisation of sensor kinase by CD spectroscopy: golden rules and tips Giuliano Siligardi , Charlotte S. Hughes , Rohanah Hussain Biochemical Society Transactions 3 DOI: 10.1042/BST20180222 Diamond Proposal Number(s): [14484, 8084, 9786, 10484] Show Abstract ▼ Abstract: This is a review that describes the golden rules and tips on how to characterise the molecular interactions of membrane sensor kinase proteins with ligands using mainly circular dichroism (CD) spectroscopy. CD spectroscopy is essential for this task as any conformational change observed in the far-UV (secondary structures (α-helix, β-strands, poly-proline of type II, β-turns, irregular and folding) and near-UV regions [local environment of the aromatic side-chains of amino acid residues (Phe, Tyr and Trp) and ligands (drugs) and prosthetic groups (porphyrins, cofactors and coenzymes (FMN, FAD, NAD))] upon ligand addition to the protein can be used to determine qualitatively and quantitatively ligand-binding interactions. Advantages of using CD versus other techniques will be discussed. The difference CD spectra of the protein–ligand mixtures calculated subtracting the spectra of the ligand at various molar ratios can be used to determine the type of conformational changes induced by the ligand in terms of the estimated content of the various elements of protein secondary structure. The highly collimated microbeam and high photon flux of Diamond Light Source B23 beamline for synchrotron radiation circular dichroism (SRCD) enable the use of minimal amount of membrane proteins (7.5 µg for a 0.5 mg/ml solution) for high-throughput screening. Several examples of CD titrations of membrane proteins with a variety of ligands are described herein including the protocol tips that would guide the choice of the appropriate parameters to conduct these titrations by CD/SRCD in the best possible way.	Dec 2018

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