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Solid-state NMR pulse sequences often feature fewer pulses and delays than the more common solution NMR experiments. This ostensible simplicity, however, belies the care with which experimental parameters must be determined, as solid-state NMR can be much less forgiving of improper experimental set-up. This is especially true of "semi-solid" samples, such as the phospholipid vesicles used to study membrane-associated peptides and proteins, which feature prominently in misfolding diseases. Protocols for the preparation of multilamellar vesicles for solid-state NMR studies of Aβ peptides are described, together with procedures for optimization of critical experimental parameters, such as spectral widths, delay times, and field strengths for (31)P, (2)H, and (13)C NMR spectroscopy.

Insulin-like peptide 3 (INSL3) is one of 10 members of the human relaxin-insulin superfamily of peptides. It is a peptide hormone that is expressed by fetal and postnatal testicular Leydig cells and postnatal ovarian thecal cells. It mediates testicular descent during fetal life and suppresses sperm apoptosis in adult males, whereas, in females, it causes oocyte maturation. INSL3 has also been shown to promote thyroid tumor growth and angiogenesis in human. These actions of INSL3 are mediated through its G protein-coupled receptor, RXFP2. INSL3, a two-chained peptide, binds to its receptor primarily via its B-chain, whereas elements of the A-chain are essential for receptor activation. In an attempt to design a high-affinity antagonist with potential clinical application as an anticancer agent as well as a contraceptive, we have previously prepared a synthetic parallel dimer of INSL3 B-chain and demonstrated that it binds to RXFP2 with high affinity. In this work, we undertook full pharmacological characterization of this peptide and show that it can antaogonize INSL3-mediated cAMP signaling through RXFP2. Further refinement by truncation of 18 residues yielded a minimized analogue that retained full binding affinity and INSL3 antagonism. It is an attractive lead peptide for in vivo evaluation as an inhibitor of male and female fertility and of INSL3-mediated carcinogenesis., (Copyright © 2010 Wiley Periodicals, Inc.)

Although the N terminus of the prion protein (PrP(C)) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23-110) and N2 (23-89) fragments derived from constitutive processing of PrP(C) and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed.

Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichiacoli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5muM), indicating a surface-associated state without significant perturbation in membrane structure. At 10muM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20muM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

NMR relaxation and diffusion characterization of several protic ionic liquids at high and low magnetic fields are reported. The dynamics of cations and anions were similar at both frequencies, with similar trends and magnitudes for a fixed component paired with oppositely charged species. An Arrhenius relationship was displayed between the molecular motion and the glass transition temperature. The diffusion of ions showed a strong degree of ion correlation between cation and anion, and Arrhenius plots of relaxation and diffusion indicated that the ions diffused as a pair. At high field diffusion was dominated by mobile species that followed Stokes-Einstein behavior. Conversely, diffusion observed at low field emphasized relatively immobile species that displayed fractional Stokes-Einstein behavior. No evidence was found to indicate the influence of magnetic field on structural and dynamic properties of the studied ILs; however, variation between diffusion coefficients at different magnetic fields indicated dynamic heterogeneities (or temporal aggregates) within the ionic liquid.

We report a novel class of self-assembling peptide nanoparticles formed by mixing aqueous solutions of K(16) peptide and a 20 amino acid peptide of net charge -5 (GLFEALLELLESLWELLLEA). Particle formation is salt-dependent and yields perfectly spherical nanoparticles of approximately 120 to approximately 800 nm diameter, depending on buffer composition and temperature, with a stoichiometry of approximately 1:2.5 for the cationic and anionic peptides. The anionic peptide forms an alpha-helix in aqueous solution, has all five glutamates on one side of the helix, and exists entirely as a discrete oligomer of 9-10 peptides. A rigid oligomer with 45-50 negative charges almost certainly represents the core component of these nanoparticles, held together by electrostatic interactions with the unstructured K(16) peptide. Cells internalize these particles by an endocytic process, and free particles are frequently seen in the cytosol, presumably because of the acid-dependent fusogenic properties of the anionic peptide. Among other applications, these particles have potential for the targeted delivery of single or multiple therapeutic moieties directly to the cytosol, and we report the successful delivery of a K(16)-linked pro-apoptosis peptide.

The interaction with model membranes of a peptide, EqtII(1-32), corresponding to the N-terminal region of the pore-forming toxin equinatoxin II (EqtII) has been studied using solid-state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [(19)F-para]Phe16-[1-(13)C]Leu19 and [(19)F-para]Phe16-[(15)N]Leu23 analogs of EqtII(1-32) measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16-23, in the presence of bilayers. (19)F-(31)P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII(1-32) in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII(1-32) in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC-bound peptide adopting helical formations at residues 16-24, whereas the DMPC:SM-bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Low-molecular-weight Brønsted acids and amine bases were used to reproducibly prepare very dry, high-purity room-temperature protic ionic liquids (PILs). A series of eight amine bases and six Brønsted acids were combined to produce 48 mixtures, of which 18 were liquid at room temperature. The phase transitions and thermal decomposition temperatures were determined for each mixture; whereas viscosity, density and conductivity were determined for the room-temperature liquids. By utilising (15)N NMR it was possible to distinguish between neutral and ionised amine bases (ammonia vs. ammonium-type ion), which indicated that the protic ionic liquids were completely ionised when made as a stoichiometric mixture. However, a Walden plot comparison of fluidity and molar conductivity indicated the majority of PILs had much lower conductivity than predicted by viscosity unless the base contained excess proton-donating groups. This disparity is indicative of protic ionic molecules forming neutral aggregates or non-Newtonian fluid hydrogen-bonded networks with a secondary Grotthuss proton-hopping mechanism arising from polyprotic bases.

Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or "rafts" in PC membranes, (31)P and (2)H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T(2) relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption., (Copyright 2009 Elsevier B.V. All rights reserved.)

Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central alpha-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the alpha-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher alpha-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.

The solution structure of fallaxidin 4.1a, a C-terminal amidated analogue of fallaxidin 4.1, a cationic antimicrobial peptide isolated from the amphibian Litoria fallax, has been determined by nuclear magnetic resonance (NMR). In zwitterionic dodecylphosphocholine (DPC) micelles, fallaxidin 4.1a adopted a partially helical structure with random coil characteristics. The flexibility of the structure may enhance the binding and penetration upon interaction with microbial membranes. Solid-state (31)P and (2)H NMR was used to investigate the effects of fallaxidin 4.1a on the dynamics of phospholipid membranes, using acyl chain deuterated zwitterionic dimyristoylphosphatidylcholine (DMPC-d(54)) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In DMPC-d(54) vesicle bilayers, fallaxidin 4.1a caused a decrease in the (31)P chemical shift anisotropy (CSA), and a decrease in deuterium order parameters from the upper acyl chain region, indicating increased lipid motion about the phosphate headgroups. Conversely, for DMPC-d(54)/DMPG, two (31)P CSA were observed due to a lateral phase separation of the two lipids and/or differing headgroup orientations in the presence of fallaxidin 4.1a, with a preferential interaction with DMPG. Little effect on the deuterated acyl chain order parameters was observed in the d(54)-DMPC/DMPG model membranes. Real time quartz crystal microbalance analyses of fallaxidin 4.1a addition to DMPC and DMPC/DMPG supported lipid bilayers together with the NMR results indicated transmembrane pore formation in DMPC/DMPG membranes and peptide insertion followed by disruption at a threshold concentration in DMPC membranes. The different interactions observed with "mammalian" (DMPC) and "bacterial" (DMPC/DMPG) model membranes imply fallaxidin 4.1a may be a useful antimicrobial peptide, with preferential cytolytic activity toward prokaryotic organisms at low peptide concentrations (<5 microM).

The reversed cubic phases (Q(II)) are a class of self-assembled amphiphile-water structures that are rich in diversity and structural complexity. These nanostructured liquid crystalline materials are generating much interest owing to their unique surface morphology, biological relevance and potential technological and medical applications. The structure of Q(II) phases in binary amphiphile-water systems is affected by the molecular structure of surfactant, water content, temperature and pressure. The presence of additives also plays an important role. The structure and phase behaviour of ternary Q(II) phases, which are comprised of two miscible amphiphiles and water, significantly differ from the binary system alone. The modulation of the phase behaviour through the addition of a second amphiphile offers an opportunity to control the size and shape of the nanostructures using a 'bottom-up' approach. In this mini-review, we discuss the structure of reversed cubic phases of amphiphile-water systems and highlight the modulation of cubic-phase structure in ternary-phase systems. We also extend this review to bulk cubic phases and the corresponding nanoscale dispersions, cubic-phase nanoparticles.

Equinatoxin II (EqtII) is a protein toxin that lyses both red blood cells and artificial membranes. Lysis is dependent on the lipid composition, with small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC) and sphingomyelin (SM) (1:1 molar) being lysed more readily than those of phosphatidylcholine alone. Removing the N-terminus of EqtII prevents pore formation, but does not prevent membrane binding. A peptide corresponding to residues 1-32 of EqtII was found using NMR to adopt a helical structure in micelles. To further understand the structural changes that accompany membrane insertion, synchrotron radiation circular dichroism spectra of the N-terminal peptide in a range of model membranes have been analysed. The peptide structure was examined in water, dodecylphosphocholine (DPC) and DPC:SM (5:1) micelles, and SUVs composed of dioleoylphosphatidylcholine (DOPC) or DMPC, together with SM and cholesterol (Chol). The peptide adopted different conformations in different lipids. Although the presence of SM did not affect the conformation in micelles, inclusion of SM in the bilayer-forming lipid increased the helicity of the peptide. This effect was abolished when Chol was added in DOPC but not in DMPC, which may relate to liquid ordered versus disordered phase properties of the lipid. SM may act as a promoter of membrane organisation necessary for membrane lysis by EqtII.

The membrane interactions of four antimicrobial peptides, aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, isolated from Australian tree frogs, are reviewed. All four peptides are amphipathic alpha-helices with a net positive charge and range in length from 13 to 25 residues. Despite several similar sequence characteristics, these peptides compromise the integrity of model membrane bilayers via different mechanisms; the shorter peptides exhibit a surface interaction mechanism while the longer peptides may form pores in membranes.

The use of radioisotope-labeled peptides and proteins in bioassays is diminishing as a consequence of the emergence of fluorescent lanthanide-labeled probes. We describe a simple, single-step labeling of synthetic human insulin-like peptide 3 (INSL3) with a commercially available activated europium chelator. The labeled peptide was then used for the development of a robust and highly reproducible receptor-binding assay which is amenable to automated high-throughput screening of ligands. Europium-labeled INSL3 showed a similar binding profile to radioactively labeled INSL3.

Liposomes were used to encapsulate rifampicin (RIF) as an alternative formulation for delivery to the respiratory tract. Factors affecting the stability of liposomes containing RIF were determined. Four liposome suspensions were prepared, containing different millimole ratios of cholesterol (CH) and soybean L-infinity-phosphatidylcholine (SPC) by the chloroform film method, followed by freeze-drying. Cryo-transmission electron microscopy, photon correlation spectroscopy, (2)H and (31)P solid-state nuclear magnetic resonance were used to characterize the liposome suspensions. Differential scanning calorimetry and X-ray diffraction were used to examine the properties of the powder formulations. The powder was dispersed through an Andersen cascade impactor to evaluate the performance of the aerosolized powder. The liposomes were a mixture of 200-300 nm unilamellar and multilamellar vesicles. Higher CH content in the liposome formulation resulted in a smaller change in size distribution with time, and higher CH content was associated with an increase in the (2)H NMR splitting, indicative of an increase in order of the lipid acyl chains. Furthermore, the SS-NMR results indicated that RIF was located between the acyl chains of the phospholipid bilayer and associated with CH molecules. Fifty percent encapsulation of RIF was obtained when the lipid content was high (SPC 10 mM: CH 10 mM). Mannitol was found to be a suitable cryoprotectant, which is attributed to its crystallinity, and use of mannitol gave particles with a mass median aerodynamic diameter of less than 5 microm. In terms of chemical stability, RIF in dry powder formulations was considerably more stable when compared to RIF aqueous solutions and RIF liposomal suspensions.

The identification in the 1950s of insulin, an essential carbohydrate regulatory hormone, as consisting of not one but two peptide chains linked by three disulfide bonds in a distinctive pattern was a milestone in peptide chemistry. When it was later found that relaxin also possessed a similar overall structure, the term 'insulin superfamily' was coined. Use of methods of conventional protein chemistry followed by recombinant DNA and more recently bioinformatics has led to the recognition that insulin is the precursor to a large protein superfamily that extends beyond the human. Insulin-like peptides are found not only in vertebrates such as mammals, birds, reptiles, amphibians but also in the invertebrates such as chordates, molluscs and insects. All superfamily members share the distinctive insulin structural motif. In the human, there exists ten members of the superfamily, each of which are expressed on the ribosome as a single-chain pre-prohormone that undergoes proteolytic processing to produce eight double-chain mature proteins and two single-chain forms. The six cysteine residues that form the three insulin disulfide cross-links - one intramolecular within the A-chain and two intermolecular between that A- and B-chains - are absolutely conserved across all members of the superfamily. They are responsible for imparting a similar overall tertiary structure. The human insulin superfamily members have each evolved to assume remarkably distinctive biological functions ranging from glucose homeostasis to neuroendocrine actions. That such diversity is contained within a modestly sized superfamily is testament to efficiency of the insulin structural motif as an evolutionary template.

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.

Skin secretions of numerous Australian tree frogs contain antimicrobial peptides that form part of the host defense mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al. (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as novel antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1, which exist as random coil in aqueous solution but have alpha-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC ( d 54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid headgroups by deuterium and (31)P NMR, respectively. In this report we demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterized using surface plasmon resonance (SPR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides has significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface interaction for aurein 1.2 and pore formation rather than membrane lysis by the longer peptides.

An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies.

Fish antifreeze proteins and glycoproteins (AF(G)Ps) prevent ice crystal growth and are able to protect mammalian cells and tissues from hypothermic damage in the sub-zero Polar oceans. This protective mechanism is not fully understood, and further data is required to explain how AF(G)Ps are able to stabilize lipid membranes as they pass through their phase transition temperatures. Solid-state NMR spectroscopy was used as a direct method to study the interaction of the 37-residue alpha-helical type I AFP, TTTT, and the low molecular weight fraction glycoprotein, AFGP8, with dimyristoylphosphatidylcholine membranes above and below the gel-fluid phase transition temperature. In contrast to previous studies in fluid phase bilayers these experiments have provided direct information regarding both the mobility of the phosphate headgroups and perturbation of the acyl chains at a range of temperatures under identical conditions on the same sample. At 5 degrees C changes in the 2H and 31P spectra and a dramatic increase in the 31P T1 relaxation times were consistent with a significant disruption of the membrane by TTTT. Heating to 30 degrees C appeared to expel the peptide from the lipid and re-cooling showed that the interaction of TTTT was not reversible. By contrast, 31P spectra of the membranes with AFGP8 were consistent with interaction with the phosphate headgroups at both 5 and 30 degrees C. Although both peptides interact with the phospholipid bilayer surface, which may stabilize the membrane at lower temperatures, the longer 31P T1 values and the 2H NMR data obtained for TTTT compared with AFGP8 suggest that TTTT causes a greater reduction of phosphate headgroup mobility and has a greater effect on the lipid acyl chains at 5 degrees C.

Since their discovery by Bangham and coworkers almost four decades ago, liposomes have become models for biomembranes and vehicles for pharmaceutical, diagnostic, and cosmetic agents. One of the advantages of using liposomes as a drug vehicle is their ability for slow release, thus reducing dosage, localizing a drug, and minimizing its side-effects. Antibiotic resistance is a growing global problem, including for ocular bacterial infection by Staphylococcus aureus, where time is an important parameter that determines the severity of infection. This situation has prompted the pursuit of ways to prepare drug-encapsulating liposomes that enable bulk release of the drug chloramphenicol (CAP) once the liposomal structure is perturbed. Our approach is a two-step process: first, to characterize the interaction of CAP with model biomembranes of dimyristoylphosphatidylcholine (DMPC) used to prepare CAP-liposomes by different formulations; second, to test the efficiency of these formulation against S. aureus. Solid-state NMR, differential scanning calorimetry, and infrared spectroscopy were used to study the interaction of CAP with DMPC bilayers. The minimum inhibitory concentrations and time-kill curves for S. aureus using different liposomal preparations were compared. Evidence of conformational changes in the DMPC molecules and the effectiveness of the CAP encapsulated in liposomes are reported.

Eighteen protic ionic liquids containing different combinations of cations and anions, hydrophobicity, viscosity, and conductivity have been synthesized and their physicochemical properties determined. In one series, the diethanolammonium cations were combined with acetate, formate, hydrogen sulfate, chloride, sulfamate, and mesylate anions. In the second series, acetate and formate anions were combined with amine bases, triethylamine, diethylamine, triethanolamine, di-n-propylamine, and di-n-butylamine. The electrochemical characteristics of the eight protic ionic liquids that are liquid at room temperature (RTPILs) have been determined using cyclic, microelectrode, and rotating disk electrode voltammetries. Potential windows of the RTPILs have been compared at glassy carbon, platinum, gold, and boron-doped diamond electrodes and generally found to be the largest in the case of glassy carbon. The voltammetry of IUPAC recommended potential scale reference systems, ferrocene/ferrocenium and cobaltocenium/cobaltocene, have been evaluated and found to be ideal in the case of the less viscous RTPILs but involve adsorption in the highly viscous ones. Other properties such as diffusion coefficients, ionic conductivity, and double layer capacitance also have been measured. The influence of water on the potential windows, viscosity, and diffusion has been studied systematically by deliberate addition of water to the dried ionic liquids. The survey highlights the problems with voltammetric studies in highly viscous room temperature protic ionic liquids and also suggests the way forward with respect to their possible industrial use.

A beta (1-42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of dementia. We detail results of 31P and 2H solid-state NMR studies of model membranes with A beta peptides and the effect of metal ions (Cu2+ and Zn2+), which are found concentrated in amyloid plaques. The effects on the lipid bilayer and the peptide structure are different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induce formation of smaller vesicles, but not when A beta(1-42) is associated with the bilayer membrane. A beta (25-35), a fragment from the C-terminal end of A beta(1-42), which lacks the metal coordinating sites found in the full length peptide, is neurotoxic to cortical cortex cell cultures. Addition of metal ions has little effect on membrane bilayers with A beta (25-35) peptides. 31P magic angle spinning NMR data show that A beta (1-42) and A beta (1-42)-Cu2+ complexes interact at the surface of anionic phospholipid membranes. Incorporated peptides, however, appear to disrupt the membrane more severely than associated peptides. Solid-state 13C NMR was used to compare structural changes of A beta (1-42) to those of A beta (25-35) in model membrane systems of anionic phospholipids and cholesterol. The A beta peptides appeared to have an increase in beta-strand structure at the C-terminus when added to phospholipid liposomes. The inclusion of Cu2+ also influenced the observed chemical shift of residues from the C-terminal half, providing structural clues for the lipid-associated A beta/metal complex. The results point to the complex pathway(s) for toxicity of the full-length peptide.

The beta-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-beta peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, A beta(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state 31P and 2H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in beta-sheet, were observed using specifically 13C-labelled A beta(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely 15N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric beta-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.

The skin secretions of Australian tree frogs are rich in peptides with potential antimicrobial activity. They interrupt bacterial cell membranes, although precisely how and whether all peptides have the same mechanism is not known. The interactions of three of these peptides-aurein 1.2, maculatin 1.1, and caerin 1.1 with supported phospholipid bilayers-are examined here using quartz crystal microbalance and atomic force microscopy. These approaches enabled us to reveal variations in material structure and density as a function of distance from the sensor surface when comparing mass sensorgrams over a range of harmonics of the natural resonance of the sensor crystal and hence obtain for the first time to our knowledge a mechanistic assessment of membrane disruption. We found that caerin inserted into the bilayer in a transmembrane manner, regardless of concentration and phospholipid composition consistent with a pore-forming mechanism. In contrast, maculatin and aurein interacted with membranes in a concentration-dependent manner. At low concentrations (<5 microM), maculatin exhibited transmembrane incorporation whereas aurein was limited to surface association. Upon reaching a threshold value of concentration, both peptides lysed the membrane. In the case of maculatin, the lysis progressed in a slow, concentration-dependent manner, forming mixed micelles, as shown by atomic force microscopy imaging. Aurein-induced lysis proceeded to a sudden disruption, which is consistent with the "carpet" mechanism. Both maculatin and aurein exhibit specificity toward phospholipids and thus have potential as candidates as antimicrobial drugs.

Solid state (13)C NMR studies of the extracellular glucans from the fungi Acremonium persicinum C38 (QM107a) and Acremonium sp. strain C106 indicated a backbone of (1-->3)-beta-linked glucosyl residues with single (1-->6)-beta-linked glucosyl side branches for both glucans. Analyses of enzymatic digestion products suggested that the average branching frequency for the A. persicinum glucan (66.7% branched) was much higher than that of the Acremonium sp. strain C106 glucan (28.6% branched). The solid state (13)C NMR spectra also indicated that both glucans are amorphous polymers with no crystalline regions, and the individual chains are probably arranged as triple helices.

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into alpha-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D(2)O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1>aurein 1.2>citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1>aurein 1.2 congruent with citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.

Abeta(1-42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Abeta aggregation and amyloid plaque formation. Abeta(25-35), a fragment from the C-terminal end of Abeta(1-42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Abeta(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Abeta(1-42). When added after vesicle formation, Abeta(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Abeta(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Abeta(25-35) retained the same beta-sheet structure irrespective of the mode of addition, the longer Abeta(1-42) appeared to have an increase in beta-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Abeta(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.

A new approach to rotational-echo double-resonance (REDOR) data analysis, analogous to Boltzmann maximum entropy statistics, is reported. This Boltzmann statistics REDOR (BS-REDOR) approach is useful for reconstructing an unbiased internuclear distance distribution for multiple internuclear distances from experimentally limited REDOR data sets on isolated spin pairs. The analysis is characterized by exploring reconstructions on model data and applied to both [1-(13)C,15N]-glycine and a long intramolecular distance in Abeta (16-22) peptide nanotubes. The approach also provides insight into the minimal number of REDOR data points required to allow faithful determination of dipolar couplings in systems with multiple internuclear distances.

The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-beta peptides Abeta40 and Abeta42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Abeta. Primarily unstructured Abeta has been shown to undergo a membrane-induced conformational change to either primarily beta-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Abeta renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Abeta peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Abeta. Here, we have attempted incorporation of Abeta in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Abeta peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Abeta40 and Abeta42 are almost entirely helical, the Abeta analogs were beta-structured in the resulting vesicle dispersions. When Abeta40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular "single channel-like" conductance of Abeta was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Abeta40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Abeta40 was present primarily as beta-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Abeta40 assemblies.

The three-dimensional structure of acetylated virginiamycin M(1) (acetylated VM1) in chloroform and in a water/acetonitrile mixture (83:17 v/v) have been established through 2D high resolution NMR experiments and molecular dynamics modeling and the results compared with the conformation of the antibiotic VM1 in the same and other solvents. The results indicated that acetylation of the C-14 OH group of VM1 caused it to rotate about 90 degrees from the position it assumed in non-acetylated VM1. The conformation of both VM1 and acetylated VM1 appear to flatten in moving from a nonpolar to polar solvent. However, the acetylated form has a more hydrophobic nature. The acetylated VM1 in chloroform and in water/acetonitrile solution had a similar configuration to that of VM1 bound to 50S ribosomes and to the Vat(D) active sites as previously determined by X-ray crystallography. Docking studies of VM1 to the 50S ribosomal binding site and the Vat(D) gave conformations very similar to those derived from X-ray crystallographic studies. The docking studies with acetylated VM1 suggested the possibility of a hydrogen bond from the acetyl carbonyl group oxygen of acetylated VM1 to the 2' hydroxyl group of ribose of adenosine 2538 at the ribosomal VM1 binding site. No hydrogen bonds between acetylated VM1 and the Vat(D) active sites were found; the loss of this binding interaction partly accounts for the release of the product from the active site.

Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.

Plasmodium falciparum develops within the mature RBCs (red blood cells) of its human host in a PV (parasitophorous vacuole) that separates the host cell cytoplasm from the parasite surface. The pore-forming toxin, SLO (streptolysin O), binds to cholesterol-containing membranes and can be used to selectively permeabilize the host cell membrane while leaving the PV membrane intact. We found that in mixtures of infected and uninfected RBCs, SLO preferentially lyses uninfected RBCs rather than infected RBCs, presumably because of differences in cholesterol content of the limiting membrane. This provides a means of generating pure preparations of viable ring stage infected RBCs. As an alternative permeabilizing agent we have characterized EqtII (equinatoxin II), a eukaryotic pore-forming toxin that binds preferentially to sphingomyelin-containing membranes. EqtII lyses the limiting membrane of infected and uninfected RBCs with similar efficiency but does not disrupt the PV membrane. It generates pores of up to 100 nm, which allow entry of antibodies for immunofluorescence and immunogold labelling. The present study provides novel tools for the analysis of this important human pathogen and highlights differences between Plasmodium-infected and uninfected RBCs.

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix-hinge-helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic alpha-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.

Amyloid-beta peptide (Abeta) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Abeta-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Abeta-Cu2+ complex to cultured primary cortical neurons was attenuated when either the pi -or tau-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Abeta and Abeta-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Abeta toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

We have studied the thermodynamic, surface, and structural properties of alphaM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of -7.2 kcal/mol. A cross-sectional molecular area of 210 A(2)/molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl(4), respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoyl-phosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyl-oleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.

Beta-amyloid peptide (Abeta), which is cleaved from the larger trans-membrane amyloid precursor protein, is found deposited in the brain of patients suffering from Alzheimer's disease and is linked with neurotoxicity. We report the results of studies of Abeta1-42 and the effect of metal ions (Cu2+ and Zn2+) on model membranes using 31P and 2H solid-state NMR, fluorescence and Langmuir Blodgett monolayer methods. Both the peptide and metal ions interact with the phospholipid headgroups and the effects on the lipid bilayer and the peptide structure were different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induced formation of smaller vesicles but when Abeta1-42 was associated with the bilayer membrane copper did not have this effect. Circular dichroism spectroscopy indicated that Abeta1-42 adopted more beta-sheet structure when incorporated in a lipid bilayer in comparison to the associated peptide, which was largely unstructured. Incorporated peptides appear to disrupt the membrane more severely than associated peptides, which may have implications for the role of Abeta in disease states.

The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.

Alzheimer's disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid beta-peptide (Abeta). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H(2)O(2)). The generation of H(2)O(2) has been linked with Abeta neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of Abeta. This residue has also been shown to be important to Abeta aggregation and amyloid formation. It is possible that the formation of an Abeta tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in Abeta aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of Abeta dimers containing dityrosine. Here we report the use of horseradish peroxidase and H(2)O(2) to dimerise N-acetyl-L-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected Abeta peptides. We also report a simple fluorescent plate reader method for monitoring Abeta dimerisation via dityrosine formation.

Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the so-called carpet model and the pore-forming model. We report here on the lytic activity of antibiotic peptides from Australian tree frogs, maculatin 1.1, citropin 1.1, and aurein 1.2, on POPC or POPC/POPG model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide/lipid mol ratio necessary to induce 50% of probe leakage was smaller for maculatin compared with aurein or citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside of single giant unilamellar vesicles is observed after peptide exposure. We conclude that maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight markers). Conversely, citropin and aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, to study the relevance of the proline residue on the membrane-action properties of maculatin, the same experimental approach was used for maculatin-Ala and maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide/lipid mol ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of maculatin-Ala and maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring maculatin sequence. As observed for maculatin, the lytic action of Maculatin-Ala and maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.

MCM-48 was surface modified via vapor-phase reactions with hexamethyldisilazane (CH(3)-MCM-48) and 3-aminopropyldimethylethoxysilane (NH(2)-MCM-48). (29)Si NMR confirmed that the resulting materials contained covalently attached trimethylsilane and 3-aminopropyldimethylsilane moieties, both important functionalities for bioseparation applications. The surface coverage was approximately 1.8 and 0.9 groups per nm(2), respectively. The X-ray diffraction patterns and the narrow pore size distributions obtained from the gas sorption isotherms showed that the modified materials retained the characteristic pore structure of the underlying MCM-48 material. CH(3)-MCM-48 exhibited significantly improved hydrolytic stability over the unmodified MCM-48 under the aqueous conditions tested, whereas NH(2)-MCM-48 appeared to be less stable than the unmodified MCM-48. The decrease in stability is most likely due to the nature of the attachment of the 3-aminopropyldimethylsilane moiety, where the conversion of surface silanol groups is limited by H bonding with the amino end, leading to a 50% lower surface concentration and resulting in an increased likelihood of nucleophilic attack on the silica surface, enhancing the rate of hydrolysis. Hexamethyldisilazane thus appears to be a superior functional group for modifying the MCM-48 surface.

The Abeta peptide has been identified as central to the onset and development of Alzheimer's disease (AD) and several hypotheses about toxicity involving Abeta peptides have been proposed including mechanisms of oxidative stress and disruption of calcium homeostasis. The biology, structure and physical properties of Abeta peptides are discussed, as well as existing therapeutics and future strategies for the treatment of AD.

The antibiotic virginiamycin is a combination of two molecules, virginiamycin M(1) (VM1) and virginiamycin S(1) (VS1) or analogues, which function synergistically by binding to bacterial ribosomes and inhibiting bacterial protein synthesis. Both VM1 and VS1 dissolve poorly in water and are soluble in more hydrophobic solvents. We have recently reported that the 3D conformation of VM1 in CDCl(3) solution differs markedly from the conformation bound to a VM1 binding enzyme and to 50S ribosomes as found by X-ray crystallographic studies. We now report the results of further NMR studies and subsequent molecular modeling of VM1 dissolved in CD(3)CN/H(2)O and compare the structure with that in CD(3)OD and CDCl(3). The conformations of VM1 in CD(3)CN/H(2)O, CD(3)OD and CDCl(3) differ substantially from one another and from the bound form, with the aqueous form most like the bound structure. We propose that the flexibility of the VM1 molecule in response to environmental conditions contributes to its effectiveness as an antibiotic.

Metal-catalysed oxidation (MCO) may play a causative role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta peptide (Abeta), the major biomarker of AD, in the presence of copper ions reduces Cu(2+) to Cu(+) and catalyses the formation of H(2)O(2) that subsequently induces radicals through Fenton chemistry. Abeta is also subject to attack by free radicals, where the presence of Cu(2+) in conjunction with H(2)O(2) catalyses oxygenation, primarily at the methionine sulfur atom. This work investigates MCO of Abeta, to gain further insight into the role of oxidative stress in AD. By combining a fluorescence assay with gel electrophoresis to monitor MCO reactions of Abeta (1-28) in the presence and absence of methionine it was determined that methionine can both protect some residues against MCO and promote the oxidation of Tyr(10) specifically. Electrospray ionization mass spectrometric analysis of methionine MCO products indicated the formation of methionine sulfoxide, methionine sulfone and related hydroxylated products. Similar products could be formed from the oxidation of Met(35) of Abeta and may relate to changes in properties of the peptide following MCO., (Copyright (c) 2004 European Peptide Society and John Wiley & Sons, Ltd.)

Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a beta-sheet conformation. One of the most abundant components in amyloid aggregates is the beta-amyloid peptide 1-42 (Abeta 1-42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Abeta 1-42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Abeta 1-42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Abeta 1-42 triggers the induction of a sequential leakage of low- and high-molecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Abeta 1-42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.

A systematic series of aluminosilicate geopolymer gels was synthesized and then analyzed using 29Si magic-angle spinning nuclear magnetic resonance (MAS NMR) in combination with Gaussian peak deconvolution to characterize the short-range ordering in terms of T-O-T bonds (where T is Al or Si). The effect of nominal Na2O/(Na2O + K2O) and Si/Al ratios on short-range network ordering was quantified by deconvolution of the 29Si MAS NMR spectra into individual Gaussian peaks representing different Q4(mAl) silicon centers. The deconvolution procedure developed in this work is applicable to other aluminosilicate gel systems. The short-range ordering observed here indicates that Loewenstein's Rule of perfect aluminum avoidance may not apply strictly to geopolymeric gels, although further analyses are required to quantify the degree of aluminum avoidance. Potassium geopolymers appeared to exhibit a more random Si/Al distribution compared to that of mixed-alkali and sodium systems. This work provides a quantitative account of the silicon and aluminum ordering in geopolymers, which is essential for extending our understanding of the mechanical strength, chemical and thermal stability, and fundamental structure of these systems.

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.