B23-Circular Dichroism
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Diamond Proposal Number(s):
[8714, 9786, 11563, 18011, 2023, 2074, 5718, 5950, 4980, 12182, 14484, 16778, 19680, 8681]
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.
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Sep 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[14986]
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.
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Aug 2019
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B23-Circular Dichroism
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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
Diamond Proposal Number(s):
[20755]
Open Access
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.
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Jul 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[13463, 11528, 14298]
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.
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May 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[12182]
Open Access
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.
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May 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[12578]
Open Access
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.
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Mar 2019
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B23-Circular Dichroism
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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
Diamond Proposal Number(s):
[15094, 16232, 17698, 19120, 20644]
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.
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Jan 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[14484, 8084, 9786, 10484]
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.
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Dec 2018
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B23-Circular Dichroism
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Christian
Bortolini
,
Tadas
Kartanas
,
Davor
Copic
,
Itzel
Condado Morales
,
Yuewen
Zhang
,
Pavan K.
Challa
,
Quentin
Peter
,
Tamas
Javorfi
,
Rohanah
Hussain
,
Mingdong
Dong
,
Giuliano
Siligardi
,
Tuomas P. J.
Knowles
,
Jerome
Charmet
Diamond Proposal Number(s):
[14750, 15005, 15592, 16203]
Abstract: Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer's and Parkinson's diseases.
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Nov 2018
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[9786, 11563, 10484]
Open Access
Abstract: Light irradiation with high photon flux in the vacuum and far-UV region is known to denature the conformation of biopolymers. Measures are in place at Diamond Light Source B23 beamline for Synchrotron Radiation Circular Dichroism (SRCD) to control and make this effect negligible. However, UV denaturation of proteins can also be exploited as a novel method for assessing biopolymer photostability as well as ligand-binding interactions. Usually, host⁻ligand binding interactions can be assessed monitoring CD changes of the host biopolymer upon ligand addition. The novel method of identifying ligand binding monitoring the change of relative rate of UV denaturation using SRCD is especially important when there are very little or insignificant secondary structure changes of the host protein upon ligand binding. The temperature study, another method used to determine molecular interactions, can often be inconclusive when the thermal effect associated with the displacement of the bound solvent molecules by the ligand is also small, making the determination of the binding interaction inconclusive. Herein we present a review on the UV-denaturation assay as a novel method to determine the relative photostability of protein formulations as well as the screening of ligand-binding interactions using the high photon flux Diamond B23 beamline for SRCD.
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Jul 2018
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