Insight into the Mechanism of Action and Peptide-Membrane Interactions of Aib-Rich Peptides: Multitechnique Experimental and Theoretical Analysis.

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib-rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane-embedded peptides. The protocols presented in this paper open new ground by showing how chiro-optical spectroscopies can throw light on the mechanism of action of AMPs.

CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups.

Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris, it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology.

Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission.

Infrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high water absorbance in the amide I region of the spectrum, means that the path length is not accurately known, so only the shape of the band is ever considered. This throws away a dimension of information. Attenuated total reflectance (ATR) IR spectroscopy is much easier to implement than transmission IR spectroscopy and, for a given instrument and sample, gives reproducible spectra. However, the ATR-absorbance spectrum varies with sample concentration and instrument configuration, and its wavenumber dependence differs significantly from that observed in transmission spectroscopy. In this paper, we determine, for the first time, how to transform water and aqueous protein ATR spectra into the corresponding transmission spectra with appropriate spectral shapes and intensities. The approach is illustrated by application to water, concanavalin A, haemoglobin and lysozyme. The transformation is only as good as the available water refractive index data. A hybrid of literature data provides the best results. The transformation also allows the angle of incidence of an ATR crystal to be determined. This opens the way to using both spectral shape and spectra intensity for protein structure fitting.

A strained alkyne-containing bipyridine reagent; synthesis, reactivity and fluorescence properties.

We report the synthesis of a bipyridyl reagent containing a strained alkyne, which significantly restricts its flexibility. Upon strain-promoted alkyne-azide cycloaddition (SPAAC) with an azide, which does not require a Cu catalyst, the structure becomes significantly more flexible and an increase in fluorescence is observed. Upon addition of Zn(ii), the fluorescence is enhanced further. The reagent has the potential to act as a fluorescent labelling agent with azide-containing substrates, including biological molecules.

Light scattering corrections to linear dichroism spectroscopy for liposomes in shear flow using calcein fluorescence and modified Rayleigh-Gans-Debye-Mie scattering.

The interpretation of data from absorbance spectroscopy experiments of liposomes in flow systems is often complicated by the fact that there is currently no easy way to account for scattering artefacts. This has proved particularly problematic for linear dichroism (LD) spectroscopy, which may be used to determine binding modes of small molecules, peptides and proteins to liposomes if we can extract the absorbance signal from the combined absorbance/scattering experiment. Equations for a modified Rayleigh-Gans-Debye (RGD) approximation to the turbidity (scattering) LD spectrum are available in the literature though have not been implemented. This review summarises the literature and shows how it can be implemented. The implementation proceeds by first determining volume loss that occurs when a spherical liposome is subjected to flow. Calcein fluorescence can be used for this purpose since at high concentrations (> 60 mM) it has low intensity fluorescence with maxima at 525 and 563 nm whereas at low concentrations (<1 mM) the fluorescence intensity is enhanced and the band shifts to 536 nm. The scattering calculation process yields the average axis ratios of the distorted liposome ellipsoids and extent of orientation of the liposomes in flow. The scattering calculations require methods to estimate liposome integrity, volume loss, and orientation when subjected to shear stresses under flow.

Spatial positioning of EB family proteins at microtubule tips involves distinct nucleotide-dependent binding properties.

EB proteins track the ends of growing microtubules and regulate microtubule dynamics both directly and by acting as the hub of the tip-tracking network. Mammalian cells express cell type-specific combinations of three EB proteins with different cellular roles. Here, we reconstitute EB1, EB2 and EB3 tip tracking in vitro We find that all three EBs show rapid exchange at the microtubule tip and that their signal correlates to the microtubule assembly rate. However, the three signals differ in their maxima and position from the microtubule tip. Using microtubules built with nucleotide analogues and site-directed mutagenesis, we show that EB2 prefers binding to microtubule lattices containing a 1:1 mixture of different nucleotides and its distinct binding specificity is conferred by amino acid substitutions at the right-hand-side interface of the EB microtubule-binding domain with tubulin. Our data are consistent with the model that all three EB paralogues sense the nucleotide state of both ß-tubulins flanking their binding site. Their different profile of preferred binding sites contributes to occupying spatially distinct domains at the temporally evolving microtubule tip structure.

Fluorescence detected linear dichroism spectroscopy: A selective and sensitive probe for fluorophores in flow-oriented systems.

Fluorescence detection typically enhances sensitivity and selectivity for fluorescent analytes. The potential for combining fluorescence detection with flow orientation of the sample in the normal configuration of linear dichroism experiments is explored in this work by measuring the fluorescence emitted from flow-orientated DNA-bound ligands and M13 bacteriophage. Data for ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenyindole are presented. The theoretical basis of the technique is also presented for instruments running in both the fixed direct-current mode, which is the normal operation mode of circular dichroism spectropolarimeters, and also in fixed high-tension voltage mode. The role of the stray light reaching the detector that results in a spectral shape in fixed direct current mode that resembles the shape of a linear dichroism spectrum, rather than the expected reduced linear dichroism, is also explored.

Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

Multifaceted Studies of the DNA Interactions and In Vitro Cytotoxicity of Anticancer Polyaromatic Platinum(II) Complexes.

This study reports a detailed biophysical analysis of the DNA binding and cytotoxicity of six platinum complexes (PCs). They are of the type [Pt(PL )(SS-dach)]Cl2 , where PL is a polyaromatic ligand and SS-dach is 1S,2S-diaminocyclohexane. The DNA binding of these complexes was investigated using six techniques including ultraviolet and fluorescence spectroscopy, linear dichroism, synchrotron radiation circular dichroism, isothermal titration calorimetry and mass spectrometry. This portfolio of techniques has not been extensively used to study the interactions of such complexes previously; each assay provided unique insight. The in vitro cytotoxicity of these compounds was studied in ten cell lines and compared to the effects of their R,R enantiomers; activity was very high in Du145 and SJ-G2 cells, with some submicromolar IC50 values. In terms of both DNA affinity and cytotoxicity, complexes of 5,6-dimethyl-1,10-phenanthroline and 2,2'-bipyridine exhibited the greatest and least activity, respectively, suggesting that there is some correlation between DNA binding and cytotoxicity.

Redox-active and DNA-binding coordination complexes of clotrimazole.

DNA interactions of anticancer mononuclear Cu(2+), Co(2+), Zn(2+), and Ni(2+) complexes with the biologically active ligand clotrimazole (clotri) are reported. To fully characterize DNA binding modes for these complexes of the formulae [M(clotri)2Cl2]·nH2O (1-4), [M(clotri)2Br2]·nH2O (5,6), [M(clotri)3NO3]NO3·nH2O (9), and [M(clotri)3(NO3)2] (10), circular dichroism (CD) and linear dichroism (LD) spectroscopy, UV melting experiments, atomic force microscopy (AFM) and ethidium bromide (EtBr) displacement methods were used. Results indicate mixed electrostatic interactions, possibly through groove binding, that result in accretion and coiling of DNA. Electrochemical studies indicate that the Cu(2+) complex 9 readily reduces to the reactive-oxygen-species-generating Cu(+), which oxidatively damages DNA. There is a subtle correlation between log P values, calculated electrostatic potentials, and cytotoxicity of the complexes. The extent of cell-nucleus DNA-metal adduct formation in the HeLa cervix-uterine carcinoma cell line does not necessarily correlate with cytotoxicity, indicating that the nature of DNA lesions may be crucial to activity.

Jahn-Teller effects on π-stacking and stereoselectivity in the phenylethaniminopyridine tris-chelates Cu(NN')3(2+).

Optically pure phenylethaniminopyridine (S(C)-L) tris-chelates of Fe(II) and other first row transition metal systems have previously been shown to give exclusively the fac structures in the solid state. Here it is shown by powder X-ray diffraction that the complex [CuL(3)][ClO(4)](2) crystallises exclusively as the mer isomer, although--for a given absolute configuration of the ligand--of the same helicity (Δ/Λ) as that displayed by the other metal complexes. The similar ligand R(C)-L(F), which contains a peripheral (19)F spin label, gave [CuL(F)(3)][ClO(4)](2) which also adopts exclusively the mer structure in the crystal, but is shown by NMR spectroscopy to have a fac:mer ratio of 1:6 in solution at low temperature. Molecular mechanics calculations for a number of isomers and conformers are consistent with the presence of such a mixture of isomers in solution for both complexes. The origin of the difference in behaviour between Fe(II) and Cu(II) is the presence of a Jahn-Teller distortion (and the generally longer M-N bonds) in the Cu(II) complexes. This disturbs intra-ligand π-stacking, leading to the poor fac/mer stereoselectivity while leaving enantioselectivity Δ/Λ apparently unaffected.

Origins of stereoselectivity in optically pure phenylethaniminopyridine tris-chelates M(NN')3(n+) (M = Mn, Fe, Co, Ni and Zn).

One-pot reactions of 2-pyridinecarboxaldehyde, chiral phenylethanamines and Fe(II) give single diastereomer fac diimine complexes at thermodynamic equilibrium so that no chiral separations are required (d.r. > 200 : 1). The origins of this stereoselectivity are partly steric and partly a result of the presence of three sets of inter-ligand parallel-offset π-stacking interactions. Mn(II), Co(II), Co(III), Ni(II) and Zn(II) give similar fac structures, alongside the imidazole analogues for Fe(II). While most of the complexes are paramagnetic, the series of molecular structures allows us to assess the influence of the π-stacking present, and there is a strong correlation between this and the M-N bond length. Fe(II) is close to optimal. For the larger Zn(II) ion, very weak π-stacking leads to poorer measured stereoselectivity (NMR) but this is improved with increased solvent polarity. The mechanism of stereoselection is further investigated via DFT calculations, chiroptical spectroscopy and the use of synthetic probes.

Self-assembling optically pure Fe(A-B)3 chelates.

Optically pure, single diastereomer fac-tris(diimine) complexes of Fe(II) are available from a remarkably facile one-pot procedure using a range of readily available (R)-2-phenylglycinol derivatives.