α-Helical conformation in the C-terminal anchoring domains of E. coli penicillin-binding proteins 4, 5 and 6

AbstractThe E. coli low molecular mass penicillin-binding proteins (PBP's) are penicillin sensitive, enzymes involved in the terminal stages of peptidoglycan biosynthesesis. These PBP's are believed to anchor to the periplasmic face of the inner membrane via C-terminal amphiphilic α-helices but to date the only support for this hypothesis has been obtained from theoretical analysis. In this paper, the conformational behaviour of synthetic peptides corresponding to these C-terminal anchoring domains was studied as a function of solvent, pH, sodium dodecyl sulphate micelles and phospholipid (DOPC, DOPG) vesicles using circular dichroism (CD) spectroscopy. The CD data showed that in 2,2,2-trifluoroethanol or sodium dodecylsulphate, all three peptides have the capacity to form an α-helical conformation but in aqueous solution or in the presence of phospholipid vesicles only those peptides corresponding to the PBP5 and PBP6 C-termini were observed to do so. A pH dependent loss of α-helical conformation in the peptide corresponding to the PBP5 C-terminus was found to correlate with the susceptibility of PBP5 to membrane extraction. This correlation would agree with the hypothesis that an α-helical conformation is required for membrane interaction of the PBP5 C-terminal region

Chiroptical studies on brevianamide B : vibrational and electronic circular dichroism confronted

Chiroptical spectroscopy, such as electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) are highly sensitive techniques to probe molecular conformation, configuration, solvation and aggregation. Here we report the application of these techniques to study the fungal metabolite brevianamide B. Comparison of the experimental ECD and VCD spectra with the density functional theory (DFT) simulated counterparts establishes that VCD is the more reliable technique to assign absolute configuration due to the larger functional and dispersion dependence of computed ECD spectra. Despite a low amount of available material, and a relatively unusual example of using VCD carbonyl multiplets, the absolute configuration could be reliably predicted, strengthening the case for application of VCD in the study of complex natural products. Spectral and crystallographic evidence for or against the formation of a dimeric aggregate is discussed; in solution the VCD spectra strongly suggest only monomeric species are present

Helically Twisted Chiral Arrays of Gold Nanoparticles Coated with a Cholesterol Mesogen

Gold nanoparticles have been prepared and surface-functionalized with a 1:1 molar mixture of a hexylthiol ligand and a chiral mesogenic ligand consisting of a cholesterylbenzoate attached via an undecylthiol spacer. Grazing incidence X-ray diffraction showed that upon annealing a columnar liquid crystal (LC) structure develops with the nanoparticles forming strings on a regular oblique 2d lattice. Synchrotron radiation circular dichroism is substantially enhanced upon the isotropic− LC transition. In the proposed structural model, layers of Au columns rotate by a small angle relative to their neighbors, with the columns winding around a helical axis. The work demonstrates that it is possible to obtain chiral LC superstructures from nanoparticles coated with chiral mesogen without the addition of a separate LC or chiral dopants. The results provide direction in the development of plasmonic metamaterials interacting selectively with circularly polarized light

Measuring Protein Structure and Stability of Protein-Nanoparticle Systems with Synchrotron Radiation Circular Dichroism

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Interactions of GFAP with ceftriaxone and phenytoin: SRCD and molecular docking and dynamic simulation

Abstract Background GFAP is the major intermediate filament protein in mature astrocytes. Its increased expression and aggregation was firstly associated to Alexander's disease, and successively in different neurological diseases including scrapie, Alzheimer's and Creutzfeld–Jacob diseases. Recently, ceftriaxone a multi-potent β-lactam antibiotic able to overcome the blood–brain barrier, successfully eliminated the cellular toxic effects of misfolded mutated GFAP, similarly to phenytoin sodium, in a cellular model of Alexander's disease and inhibited α-synuclein aggregation protecting PC12 cells from the exposure to 6-hydroxydopamine. Methods In this study, synchrotron radiation circular dichroism spectroscopy has been used to obtain structural information about the GFAP-ceftriaxone (phenytoin) interactions, while computational methods allowed the identification of the relevant putative binding site of either ceftriaxone or phenytoin on the dimer structure of GFAP, permitting to rationalize the spectroscopic experimental results. Results We found that GFAP exhibited enhanced stability upon the addition of two equivalents of each ligands with ceftriaxone imparting a more spontaneous interactions and a more ordered complex system than phenytoin. Conclusions SRCD data and MD models indicate a stronger protective effect of ceftriaxone in neurological disorders characterized by an increased production and polymerization of GFAP. General significance This result, in addition to our previous works in which we documented that ceftriaxone interacts with α-synuclein inhibiting its pathological aggregation and that a cyclical treatment with this molecule in a patient with adult-onset Alexander's disease halted, and partly reversed, the progression of neurodegeneration, suggests the possibility of a chaperone-like effect of ceftriaxone on protein involved in specific neurodegenerative diseases

Chirality enhancement in macro-chiral liquid crystal nanoparticles

The amplification of molecular chirality by metal nanoparticles (NPs) is an important and rapidly evolving field in nanomaterial research with wide applications in smart materials, catalysis, and solvent-solute interactions. Here we present the results of the synthesis of gold nanoparticles (AuNPs) functionalized both with chiral ligands based on the binaphthol motif and with nematogenic groups (ChirAuLC). The materials were characterized chemically and the ratios between chiral groups and LC groups was determined. Synchrotron radiation circular dichroism (SRCD) and synchrotron based X-ray diffraction (XRD) studies show that the AuNPs favoured by the LC state arrange themselves into ordered columns and a helical superstructure appears in the mesophase of collective NPs. A specific focus has been the investigation of the chiral induction of ChirAuLC composites in two different nematic LC hosts. For a number of selected mixtures, the helical twisting power (HTP) of these NPs in systems was calculated from systematic optical observations based on optical polarizing microscopy (OPM). The experimental data show that the HTP of the investigated ChirAuLC composite is significantly larger than that of free "small molecule"chiral groups when dispersed in the same LC host and the chiral transfer efficiency of ChirAuLC is higher than NPs functionalized only with chiral groups (ChirAuNP). This is new and can be explained by a combination of a surface chirality and the domino effect of bound mesogens interacting with the bulk. This journal i

Graphene FET sensors for Alzheimer’s disease protein biomarker clusterin detection

We report on the fabrication and characterisation of Graphene field-effect transistor (GFET) Biosensors for detecting clusterin, a prominent protein biomarker of Alzheimer’s disease (AD). There are approximately 54 million people currently living with dementia worldwide and this is expected to rise to 130 million by 2050. Although there are over 400 different types of dementia, AD is the most common type, affecting between 50–75% of those diagnosed with dementia. Diagnosis of AD can take up to 2 years currently using MRI, PET, CT scans and memory tests. There is, therefore, an urgent need to develop low-cost, accurate, non-invasive and point-of-care (PoC) sensors for early diagnosis of AD. The GFET sensors we are developing to address this challenge were fabricated on Si/SiO2 substrate through processes of photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with a linker molecule and anti-clusterin antibody. The detection was achieved through the binding reaction between the antibody and varying concentrations of clusterin antigen from 1 pg/mL to 1 ng/mL. The GFETs were characterized using 4-probe direct current (DC) electrical measurements which demonstrated a limit of detection of the biosensors to be below 1 pg/mL

Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling

Porphyrins were attached to LNA uridine building blocks via rigid 5-acetylene or more flexible propargyl-amide linkers and incorporated into DNA strands. The systems show a greatly increased thermodynamic stability when using as little as three porphyrins in a zipper arrangement. Thermodynamic analysis reveals clustering of the strands into more ordered duplexes with both greater negative ??S and ??H values, and less ordered duplexes with small positive ??S differences, depending on the combination of linkers used. The exciton coupling between the porphyrins is dependent on the flanking DNA sequence in the single stranded form, and on the nature of the linker between the nucleobase and the porphyrin in the double stranded form; it is, however, also strongly influenced by intermolecular interactions. This system is suitable for the formation of stable helical chromophore arrays with sequence and structure dependent exciton coupling

The Secondary Structure of a Major Wine Protein is Modified upon Interaction with Polyphenols

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

Circular dichroism spectroscopy identifies the β-adrenoceptor agonist salbutamol as a direct inhibitor of tau filament formation in vitro.

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