B23-Circular Dichroism
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Diamond Proposal Number(s):
[14703]
Abstract: Twenty Apolipoprotein A-I (ApoA-I) variants are responsible for a systemic hereditary amyloidosis in which protein fibrils can accumulate in different organs, leading to their failure. Several ApoA-I amyloidogenic mutations are also associated with hypoalphalipoproteinemia, low ApoA-I and high-density lipoprotein (HDL)-cholesterol plasma levels; however, subjects affected by ApoA-I-related amyloidosis do not show a higher risk of cardiovascular diseases (CVD). The structural features, the lipid binding properties and the functionality of four ApoA-I amyloidogenic variants were therefore inspected in order to clarify the paradox observed in the clinical phenotype of the affected subjects.
Our results show that ApoA-I amyloidogenic variants are characterized by a different oligomerization pattern and that the position of the mutation in the ApoA-I sequence affects the molecular structure of the formed HDL particles. Although lipidation increases ApoA-I proteins stability, all the amyloidogenic variants analyzed show a lower affinity for lipids, both in vitro and in ex vivo mouse serum. Interestingly, the lower efficiency at forming HDL particles is compensated by a higher efficiency at catalysing cholesterol efflux from macrophages.
The decreased affinity of ApoA-I amyloidogenic variants for lipids, together with the increased efficiency in the cholesterol efflux process, could explain why, despite the unfavourable lipid profile, patients affected by ApoA-I related amyloidosis do not show a higher CVD risk.
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Sep 2017
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[13319, 12555]
Abstract: For the purpose of proteins bio-conjugation to Gold Nano-Particles (GNPs), we designed and synthesized a polypeptide named NanoLock, derived from SNARE (Soluble NSF Attachment protein REceptor) proteins and able to form a remarkably stable complex with the SNARE protein SNAP25 (Synaptosome Associated Protein of 25 kDa). We also characterized the adsorption of a SNAP25 recombinant fusion to Glutathione S-Transferases (GST) named GST-SNAP25 onto GNPs and found that it forms a stable protein corona surrounding GNPs. Using GST-SNAP25 as an intermediate protein, passively adsorbed on GNPs, we were able to stably bind NanoLock to GNPs.
By fusing an arbitrary protein of interest to the affinity tag NanoLock, it would be in principle possible to bind any protein to GST-SNAP25 coated GNPs by simple mixing, in a site-oriented way. Therefore, we propose the pair NanoLock/GST-SNAP25 as a universal tool for the easy bio-conjugation of recombinant proteins to GNPs and possibly other gold surfaces. Further engineered versions of SNAP25, able to bind surfaces other than gold, could be used to decorate with proteins other materials, taking advantage of the same modular approach of the system described here in the case of GNPs.
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Sep 2017
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[14484, 12003, 10310, 4986, 9016, 5056]
Open Access
Abstract: This data article presents the results from quality control experiments including N-terminal sequencing, SEC-MALS and Mass Spectrometry for purified VanSA used in experiments described in Hughes et. al. 2017 [1]; in addition to ligand interaction measurements and thermal melting curves of VanSA in the presence of screened ligands from circular dichroism meaurements as well as UV-Vis absorbance spectra for the binding interaction of VanSA in the presence of screened ligands.
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Jul 2017
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B23-Circular Dichroism
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Isabel
Gonçalves Silva
,
Inna M.
Yasinska
,
Svetlana S.
Sakhnevych
,
Walter
Fiedler
,
Jasmin
Wellbrock
,
Marco
Bardelli
,
Luca
Varani
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Giacomo
Ceccone
,
Steffen M.
Berger
,
Yuri A.
Ushkaryov
,
Bernhard F.
Gibbs
,
Elizaveta
Fasler-kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[12578]
Open Access
Abstract: Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.
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Jul 2017
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[14894, 16440]
Abstract: The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism, and small angle neutron scattering, we have studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered, with a mobile C-terminal domain and no changes in secondary structure but increases in the radius of gyration on complex formation. Comparison of wild type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.
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Jul 2017
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[12332]
Open Access
Abstract: T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity.
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Jul 2017
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B23-Circular Dichroism
RF
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Diamond Proposal Number(s):
[10415, 12668]
Abstract: The design and synthesis of water soluble, amino-acid-functionalised naphthalenediimides (NDIs) as potential ligands of native G-quadruplexes is reported. The NDIs were tested on a panel of oncogene promoters, on the human telomeric sequence h-telo, and on double-stranded DNA. Out of the ligands tested, NDI 3 (Nϵ-Boc-l-lysine NDI) exhibited a highly discriminating nature by only stabilising the oncogene promoter c-kit2, which is up-regulated up to 80 % in ovarian, gastrointestinal, and breast malignancies.
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Jun 2017
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[1681, 2074]
Open Access
Abstract: Previous clinical research has suggested high-affinity binding of flavopiridol (FLAP) to human blood serum proteins,
specifically either human serum albumin (HSA) or human alpha-1-acid glycoprotein (hAGP), when compared to fetal bovine
serum albumin (BSA) or bovine alpha-1-acid glycoprotein (bAGP) used in pre-clinical assays. This high-affinity binding was
suggested as the reason for its poor human clinical trial performance as a treatment for chronic lymphocytic leukaemia (CLL).
Using three biophysical techniques, specifically circular dichroism (CD), isothermal calorimetry (ITC) and fluorescence spectroscopy,
I show that FLAP does not have an overly high-affinity for either fetal BSA, HSA, bAGP or hAGP. I therefore suggest
an alternate hypothesis that models the albumin and alpha-1-acid glycoprotein (AGP) binding sites at the different protein concentrations
used in the fetal bovine pre-clinical assay and human physiological conditions. I use analytical ultracentrifugation
(AUC) experiments to determine the validity of the theoretical models. The models can also be altered to account for the elevated
AGP levels and reduced albumin levels seen in human cancer patients. Major differences in the concentrations of free
available FLAP are observed between the fetal bovine pre-clinical model and human physiological conditions. A number of
recommendations can therefore be made on how future pre-clinical assay studies should be conducted.
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Jun 2017
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[14484, 12003, 10310, 4986, 9016, 5056]
Open Access
Abstract: A-type resistance towards “last-line” glycopeptide antibiotic vancomycin in the leading hospital acquired infectious agent, the enterococci, is the most common in the UK. Resistance is regulated by the VanRASA two-component system, comprising the histidine sensor kinase VanSA and the partner response regulator VanRA. The nature of the activating ligand for VanSA has not been identified, therefore this work sought to identify and characterise ligand(s) for VanSA. In vitro approaches were used to screen the structural and activity effects of a range of potential ligands with purified VanSA protein. Of the screened ligands (glycopeptide antibiotics vancomycin and teicoplanin, and peptidoglycan components N-acetylmuramic acid, D-Ala-D-Ala and Ala-D-y-Glu-Lys-D-Ala-D-Ala) only glycopeptide antibiotics vancomycin and teicoplanin were found to bind VanSA with different affinities (vancomycin 70 μM; teicoplanin 30 and 170 μM), and were proposed to bind via exposed aromatic residues tryptophan and tyrosine. Furthermore, binding of the antibiotics induced quicker, longer-lived phosphorylation states for VanSA, proposing them as activators of type A vancomycin resistance in the enterococci.
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May 2017
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B23-Circular Dichroism
I02-Macromolecular Crystallography
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John M.
Kelly
,
Paraic
Keane
,
James P.
Hall
,
Fergus
Poynton
,
Bjorn
Poulsen
,
Sarah P.
Gurung
,
Ian P.
Clark
,
Igor
Sazanovich
,
Michael
Towrie
,
Thorfinnur
Gunnlaugsson
,
Susan J.
Quinn
,
Christine J.
Cardin
Diamond Proposal Number(s):
[9684, 11291]
Abstract: Key to the development of DNA-targeting phototherapeutic drugs is determining the interplay between the photoactivity of the drug and its binding preference for a target sequence. For the photo-oxidising lambda-[Ru(TAP)2(dppz)]2+ (Ʌ-1) complex bound to either d{T1C2G3G4C5G6C7C8G9A10}2 (G9) or d{TCGGCGCCIA}2 (I9), the X-ray crystal structures shows the dppz intercalated at the terminal T1C2;G9A10 step or T1C2;I9A10 step. Thus substitution of the G9 nucleobase by inosine does not affect intercalation in the solid state although with I9 the dppz is more deeply inserted. In solution it is found that the extent of guanine photo-oxidation, and the rate of back electron transfer, as determined by ps and ns time-resolved infrared and transient visible absorption spectroscopy, is enhanced in I9, despite it containing the less oxidisable inosine. This is attributed to the nature of the binding in the minor groove due to the absence of an NH2 group. Similar behaviour and the same binding site in the crystal.are found for d{TTGGCGCCAA}2 (A9), In solution we propose that intercalation occurs at the C2G3;C8I9 or T2G3;C8A9 steps, respectively, with G3 the likely target for photo-oxidation. This demonstrates how changes in the minor groove (in this case removal of an NH2 group) can facilitate binding of Ru(II)dppz complexes and hence influence any sensitised reactions occurring at these sites. No similar enhancement of photooxidation on binding to I9 is found for the delta enantiomer.
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May 2017
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