B23-Circular Dichroism
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Diamond Proposal Number(s):
[15313, 14658, 14430]
Abstract: Background: Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3′-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins. Methods: Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays. Results: AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed β-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14–3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins. Conclusions: This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection.
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Dec 2020
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[17645, 24138]
Abstract: Potential drug treatments for Alzheimer’s disease (AD) may be found by identifying compounds that block the assembly of the microtubule-associated protein tau into neurofibrillar tangles associated with neuron destabilisation and cell death. Here, a small library of structurally diverse compounds was screened in vitro for the ability to inhibit tau aggregation, using high-throughput synchrotron radiation circular dichroism (HT-SRCD) as a novel tool to monitor the structural changes in the protein as it assembles into filaments. The catecholamine epinephrine was found to be the most effective tau aggregation inhibitor of all 88 compounds screened. Subsequently, we tested chemically-similar phenolamine drugs from the β-adrenergic receptor (βAR) agonist class, using conventional circular dichroism spectroscopy, thioflavin T fluorescence and transmission electron microscopy. Two compounds, salbutamol and dobutamine, used widely in the treatment of respiratory and cardiovascular disease, impede the aggregation of tau in vitro. Dobutamine reduces both the rate and yield of tau filament formation over 24 hours, although it has little effect on the structural transition of tau into β-sheet structures over 24 hours. Salbutamol also reduces the yield and rate of filament formation, and additionally inhibits tau’s structural change into β-sheet rich aggregates. Salbutamol has a good safety profile and a half-life that facilitates permeation through the blood brain barrier and could represent an expediated approach to developing AD therapeutics. These results provide the motivation for in vivo evaluation of pre-existing β-adrenergic receptor agonists as a potential therapy for AD through the reduction of tau deposition in AD.
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Jun 2020
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[16501, 14084]
Abstract: Lipidic bicelles have been widely used for the analysis of integral membrane proteins where their spontaneous alignment in an magnetic field has been exploited for oriented sample solid-state NMR studies. Many of their physical properties however make them well suited to the analysis of membrane proteins by circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD). In this paper we have identified bicelle compositions that permit comparable studies of integral membrane proteins by solid-state NMR, CD, and SRCD; complementary methods that can provide insights into protein structure and the interactions with drugs and other small molecules. Furthermore we have been able to identify bicelle compositions that allow the magnetic alignment of the bicelles at fields routinely available in magnetic CD instruments. This potentially provides a route to the preparation of samples for oriented CD that mitigates many of the issues associated with the preparation of mechanically aligned samples, although we demonstrate that the dynamics present within the system can complicate the analysis of such spectra.
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May 2020
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Abstract: The effect of spin polarization in conduction and electric field-induced polarization was measured for double-stranded DNA oligonucleotides and oligopeptides of different lengths. The measurements were conducted using magnetic contact AFM, spin-dependent electrochemistry, spin-dependent polarization, and magnetoresistance studies. It was established that the spin-dependent conduction through chiral molecules depends on the voltage applied with a power of d, when d is larger than unity, and that there is a different voltage threshold for conducting each of the spin polarizations. In addition, there is no spin flipping during the conduction through the chiral system. The spin polarization depends linearly on the length, within the range of lengths studied, and it seems to scale like the optical activity. These results suggest the importance of the electric polarizability in the chiral-induced spin selectivity process.
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Apr 2020
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[14708, 16031]
Open Access
Abstract: Polyphenols are an important constituent of wines and they are largely studied due to their antioxidant properties and for their effects on wine quality and stability, which is also related to their capacity to bind to proteins. The effects of some selected polyphenols, including procyanidins B1 and B2, tannic acid, quercetin, and rutin, as well as those of a total white wine procyanidin extract on the conformational properties of the major wine protein VVTL1 (Vitis vinifera Thaumatin-Like-1) were investigated by Synchrotron Radiation Circular Dichroism (SRCD). Results showed that VVTL1 interacts with polyphenols as demonstrated by the changes in the secondary (far-UV) and tertiary (near-UV) structures, which were differently affected by different polyphenols. Additionally, polyphenols modified the two melting temperatures (TM) that were found for VVTL1 (32.2 °C and 53.9 °C for the protein alone). The circular dichroism (CD) spectra in the near-UV region revealed an involvement of the aromatic side-chains of the protein in the interaction with phenolics. The data demonstrate the existence of an interaction between polyphenols and VVTL1, which results in modification of its thermal and UV denaturation pattern. This information can be useful in understanding the behavior of wine proteins in presence of polyphenols, thus giving new insights on the phenomena that are involved in wine stability.
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Apr 2020
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[20210, 21575]
Abstract: The eye lens is a biconvex structure composed of lens fibres, cells that lack of blood and nerve supply and of any organelle, allowing for a high concentration of water soluble proteins that determine the lens transparency and refractive index. The lens water soluble protein pool in mammals is composed of α‐, β‐, and γ‐crystallins, the latter being involved in calcium homeostasis and having structural importance, the first playing a crucial role in preventing protein aggregation and the consequent lens obfuscation, which leads to the clinical outcome of cataract. Among different factors, oxidative stress, free radicals, and reactive oxygen species (ROSs) generated by the exposure to UV light are widely recognized to cause cataract formation. Taking advantage of synchrotron radiation circular dichroism, fluorescence, and circular dichroism spectroscopies, in the present study we investigate the influence of different small molecules with the potential to either quench ROS generation or to stabilize protein conformation. Therefore, ascorbic acid, an excellent antioxidant agent already present in the eye aqueous humour, has been tested along with ceftriaxone, mannitol and trehalose, which osmolyte activity was demonstrated interfering with model proteins misfolding. Our results showed that ascorbic acid strongly inhibits the ROS production without, however, preserving the native protein structure, whereas mannitol had no effect on the ROS production but retained better the secondary structure of WS proteins. Collectively, the use of a mixture of ascorbic acid and mannitol could be used to better protect eye lens proteins from ROS damage preventing the cataract onset.
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Mar 2020
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B23-Circular Dichroism
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Open Access
Abstract: Light-harvesting complex II (LHCII) from the marine green macroalga Bryopsis corticulans is spectroscopically characterized to understand the structural and functional changes resulting from adaptation to intertidal environment. LHCII is homologous to its counterpart in land plants but has a different carotenoid and chlorophyll (Chl) composition. This is reflected in the steady-state absorption, fluorescence, linear dichroism, circular dichroism and anisotropic circular dichroism spectra. Time-resolved fluorescence and two-dimensional electronic spectroscopy were used to investigate the consequences of this adaptive change in the pigment composition on the excited-state dynamics. The complex contains additional Chl b spectral forms – absorbing at around 650 nm and 658 nm – and lacks the red-most Chl a forms compared with higher-plant LHCII. Similar to plant LHCII, energy transfer between Chls occurs on timescales from under hundred fs (mainly from Chl b to Chl a) to several picoseconds (mainly between Chl a pools). However, the presence of long-lived, weakly coupled Chl b and Chl a states leads to slower exciton equilibration in LHCII from B. corticulans. The finding demonstrates a trade-off between the enhanced absorption of blue-green light and the excitation migration time. However, the adaptive change does not result in a significant drop in the overall photochemical efficiency of Photosystem II. These results show that LHCII is a robust adaptable system whose spectral properties can be tuned to the environment for optimal light harvesting.
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Mar 2020
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[8034, 20210]
Open Access
Abstract: The eye lens is mainly composed of the highly ordered water-soluble (WS) proteins named crystallins. The aggregation and insolubilization of these proteins lead to progressive lens opacification until cataract onset. Although this is a well-known disease, the mechanism of eye lens protein aggregation is not well understood; however, one of the recognized causes of proteins modification is related to the exposure to UV light. For this reason, the spectroscopic properties of WS lens proteins and their stability to UV irradiation have been evaluated by different biophysical methods including synchrotron radiation circular dichroism, fluorescence, and circular dichroism spectroscopies. Moreover, dynamic light scattering, gel electrophoresis, transmission electron microscopy, and protein digestion followed by tandem LC–MS/MS analysis were used to study the morphological and structural changes in protein aggregates induced by exposure to UV light. Our results clearly indicated that the exposure to UV radiation modified the protein conformation, inducing a loss of ordered structure and aggregation. Furthermore, we confirmed that these changes were attributable to the generation of reactive oxygen species due to the irradiation of the protein sample. This approach, involving the photodenaturation of proteins, provides a benchmark in high-throughput screening of small molecules suitable to prevent protein denaturation and aggregation.
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Feb 2020
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B23-Circular Dichroism
I19-Small Molecule Single Crystal Diffraction
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Zongsu
Han
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Kunyu
Wang
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Yifan
Guo
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Wenjie
Chen
,
Jiale
Zhang
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Xinran
Zhang
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Giuliano
Siligardi
,
Sihai
Yang
,
Zhen
Zhou
,
Pingchuan
Sun
,
Wei
Shi
,
Peng
Cheng
Diamond Proposal Number(s):
[20293]
Open Access
Abstract: The integration of luminescence and chirality in easy-scalable metal-organic frameworks gives rise to the development of advanced luminescent sensors. To date, the synthesis of chiral metal-organic frameworks is poorly predictable and their chirality primarily originates from components that constitute the frameworks. By contrast, the introduction of chirality into the pores of metal-organic frameworks has not been explored to the best of our knowledge. Here, we demonstrate that chirality can be introduced into an anionic Zn-based metal-organic framework via simple cation exchange, yielding dual luminescent centers comprised of the ligand and Tb3+ ions, accompanied by a chiral center in the pores. This bifunctional material shows enantioselectivity luminescent sensing for a mixture of stereoisomers, demonstrated for Cinchonine and Cinchonidine epimers and amino alcohol enantiomers, from which the quantitative determination of the stereoisomeric excess has been obtained. This study paves a pathway for the design of multifunctional metal-organic framework systems as a useful method for rapid sensing of chiral molecules.
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Nov 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[12182, 14484, 16778, 19680]
Open Access
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.
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Nov 2019
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