Phase evolution of Na2O-Al2O3-SiO2-H2O gels in synthetic aluminosilicate binders.

This study demonstrates the production of stoichiometrically controlled alkali-aluminosilicate gels ('geopolymers') via alkali-activation of high-purity synthetic amorphous aluminosilicate powders. This method provides for the first time a process by which the chemistry of aluminosilicate-based cementitious materials may be accurately simulated by pure synthetic systems, allowing elucidation of physicochemical phenomena controlling alkali-aluminosilicate gel formation which has until now been impeded by the inability to isolate and control key variables. Phase evolution and nanostructural development of these materials are examined using advanced characterisation techniques, including solid state MAS NMR spectroscopy probing (29)Si, (27)Al and (23)Na nuclei. Gel stoichiometry and the reaction kinetics which control phase evolution are shown to be strongly dependent on the chemical composition of the reaction mix, while the main reaction product is a Na2O-Al2O3-SiO2-H2O type gel comprised of aluminium and silicon tetrahedra linked via oxygen bridges, with sodium taking on a charge balancing function. The alkali-aluminosilicate gels produced in this study constitute a chemically simplified model system which provides a novel research tool for the study of phase evolution and microstructural development in these systems. Novel insight of physicochemical phenomena governing geopolymer gel formation suggests that intricate control over time-dependent geopolymer physical properties can be attained through a careful precursor mix design. Chemical composition of the main N-A-S-H type gel reaction product as well as the reaction kinetics governing its formation are closely related to the Si/Al ratio of the precursor, with increased Al content leading to an increased rate of reaction and a decreased Si/Al ratio in the N-A-S-H type gel. This has significant implications for geopolymer mix design for industrial applications.

A routine method for cloning, expressing and purifying Aß(1-42) for structural NMR studies.

Nuclear magnetic resonance (NMR) is a key technology in the biophysicist's toolbox for gaining atomic-level insight into structure and dynamics of biomolecules. Investigation of the amyloid-ß peptide (Aß) of Alzheimer's disease is one area where NMR has proven useful, and holds even more potential. A barrier to realizing this potential, however, is the expense of the isotopically enriched peptide required for most NMR work. Whereas most biomolecular NMR studies employ biosynthetic methods as a very cost-effective means to obtain isotopically enriched biomolecules, this approach has proven less than straightforward for Aß. Furthermore, the notorious propensity of Aß to aggregate during purification and handling reduces yields and increases the already relatively high costs of solid phase synthesis methods. Here we report our biosynthetic and purification developments that yield pure, uniformly enriched ¹5N and ¹³C¹5N Aß(1-42), in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods, which we characterize by solution-state NMR, thioflavin T assays, circular dichroism, electrospray mass spectrometry, and dynamic light scattering. These developments should facilitate further investigations into Alzheimer's disease, and perhaps misfolding diseases in general.

Solid-state NMR structure characterization of a 13CO-Labeled Ir(I) complex with a P,N-donor ligand including ultrafast MAS methods.

The structural characterization of a (13)CO-labeled Ir(I) complex bearing an P,N-donor ligand (1-[2-(diphenylphosphino)ethyl]pyrazole), [Ir(PyP)((13)CO)Cl] is demonstrated using a series of tailored solid-state NMR techniques based on ultrafast (60 kHz) Magic Angle Spinning (MAS), which facilitates correlations with narrow proton line-widths. Our 1D (1)H MAS and 2D (13)C and (31)P CP-MAS NMR spectra provided structural information similar to that obtained using NMR spectroscopy in solution. We employed high-resolution 2D solid-state correlation spectroscopy ((1)H-(13)C HETCOR, (1)H-(31)P correlation) to characterize the networks of dipolar couplings between protons and carbon/phosphorus. (1)H-(1)H SQ-SQ correlation spectra showed the dipolar contacts between all protons in a similar fashion to its solution counterpart, NOESY. The use of the (1)H single quantum/double quantum experiments made it possible to observe the dipolar-coupling contacts between immediately adjacent protons. Additionally, internuclear (13)CO-(31)P distance measurements were performed using REDOR. The combination of all of these techniques made it possible to obtain comprehensive structural information on the molecule [Ir(PyP)((13)CO)Cl] in the solid state, which is in excellent agreement with the single crystal X-ray structure of the complex, and demonstrates the enormous value of ultrafast MAS NMR techniques for a broad range of future applications.

Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment.

A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.

Context dependence of protein misfolding and structural strains in neurodegenerative diseases.

Vast arrays of structural forms are accessible to simple amyloid peptides and environmental conditions can direct assembly into single phases. These insights are now being applied to the aggregation of the Aß peptide of Alzheimer's disease and the identification of causative phases. We extend use of the imaging agent Pittsburgh compound B to discriminate among Aß phases and begin to define conditions of relevance to the disease state. Also, we specifically highlight the development of methods for defining the structures of these more complex phases.

A practical implementation of de-Pake-ing via weighted Fourier transformation.

We provide an NMRPipe macro to meet an increasing need in membrane biophysics for facile de-Pake-ing of axially symmetric deuterium, and to an extent phosphorous, static lineshapes. The macro implements the development of McCabe & Wassall (1997), and is run as a simple replacement for the usual Fourier transform step in an NMRPipe processing procedure.

The lipid network.

Natural cell membranes are composed of a remarkable variety of lipids, which provide specific biophysical properties to support membrane protein function. An improved understanding of this complexity of membrane composition may also allow the design of membrane active drugs. Crafting a relevant model of a cell membrane with controlled composition is becoming an art, with the ability to reveal the molecular mechanisms of biological processes and lead to better treatment of pathologies. By matching physiological observations from in vivo experiments to high-resolution information, more easily obtained from in vitro studies, complex interactions at the lipid interface are determined. The role of the lipid network in biological membranes is, therefore, the subject of increasing attention.

Solid-state NMR of amyloid membrane interactions.

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 Aß 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.

Disentanglement of heterogeneous dynamics in mixed lipid systems.

Static phosphorous NMR has been a powerful technique for the study of model supramolecular phospholipid structures. Application to natural lipid bilayers with complex compositions, however, has been severely limited by the difficulty in deconvoluting overlapping broad lineshapes. We demonstrate a solution to this problem, using a global fit to a few slow magic-angle spinning spectra, in combination with an adaptation of Boltzmann statistics maximum entropy. The method provides a model-free means to characterize a heterogeneous mix of lipid dynamics via a distribution of (31)P chemical shift anisotropies. It is used here to identify clear changes in membrane dynamics of a phosphatidylethanolamine and phosphatidylglycerol mixture, mimicking an Escherichia coli membrane upon addition of just 2% of the antimicrobial peptide maculatin 1.1. This illustration opens the prospect for investigation of arbitrarily complex natural lipid systems, important in many areas of biophysical chemistry and biomedicine.

Lipid matrix plays a role in Abeta fibril kinetics and morphology.

While neuronal membranes are proposed to be the primary target of amyloid plaques, the effect of phospholipids on fibril formation kinetics and morphology has not yet been resolved. We report that interaction of various compositions with neuronal mimics promoted different processes of fibril formation: negatively charged surfaces increased the lag time and elongation rate in thioflavin T assays, while brain total lipid extract had an opposite effect compared to that in the absence of lipid. Electron microscopy showed thin and elongated fibrils when the peptide was incubated with anionic lipids, while neutral surfaces promoted coarse and small fibrils. Circular dichroism and thioflavin T assays confirmed an initially unstructured peptide, and measured its transition to an aggregated beta-sheet conformation.

Interactions of a synthetic Leu-Lys-rich antimicrobial peptide with phospholipid bilayers.

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH(2)) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the ß-sheet content to ~20%. Addition of negatively charged LUV, DMPC-dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d(54)-DMPC) and mixed d(54)-DMPC-DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC-DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by (31)P NMR. However, no perturbations in the T(1) relaxation nor the T(2-) values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).

Real-time quantitative analysis of lipid disordering by aurein 1.2 during membrane adsorption, destabilisation and lysis.

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.

Self-assembly of peptides into spherical nanoparticles for delivery of hydrophilic moieties to the cytosol.

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.

Solution structure and membrane interactions of the antimicrobial peptide fallaxidin 4.1a: an NMR and QCM study.

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).

Generalized biaxial shearing of MQMAS NMR spectra.

Two dimensional multiple quantum (MQ) MAS NMR experiments have become popular due to the wide applicability of this technique to structural questions in materials science, the abundance of half-integer spin nuclei in the periodic table, and the ease of implementation on typical solid state NMR instruments. In spite of the high-resolution theoretically possible from such experiments, the homogeneous and inhomogeneous broadening factors inherent in many samples of interest can make spectral analysis challenging. We present several possible spectral shearing schemes that may be useful for spectral analysis, and in particular we introduce shearing in the directly detected dimension. We suggest that for amorphous or disordered samples that give broad spectral features, shearing may be used as a general tool for optimal positioning of these features relative to one another and for the characterization of isotropic chemical and quadrupolar shifts.

Membrane interactions of antimicrobial peptides from Australian frogs.

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.

Chemical shift mapping of gammadelta resolvase dimer and activated tetramer: mechanistic implications for DNA strand exchange.

Several static structural models exist for gammadelta resolvase, a self-coded DNA recombinase of the gammadelta transposon. While these reports are invaluable to formulation of a mechanistic hypothesis for DNA strand exchange, several questions remain. Foremost among them concerns the protomer structural dynamics within the protein/DNA synaptosome. Solution NMR chemical shift assignments have been made for truncated variants of the natural wild-type dimer, which is inactive without the full synaptosome structure, and a mutationally activated tetramer. Of the 134 residues, backbone (1)H, (15)N, and (13)Calpha assignments are made for 121-124 residues in the dimer, but only 76-80 residues of the tetramer. These assignment differences are interpreted by comparison to X-ray diffraction models of the recombinase dimer and tetramer. Inspection of intramolecular and intermolecular structural variation between these models suggests a correspondence between sequence regions at subunit interfaces unique to tetramer, and the regions that can be sequentially assigned in the dimer but not the tetramer. The loss of sequential context for assignment is suggestive of stochastic fluctuation between structural states involving protomer-protomer interactions exclusive to the activated tetrameric state, and may be indicative of dynamics which pertain to the recombinase mechanism.

Effect of antimicrobial peptides from Australian tree frogs on anionic phospholipid membranes.

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.

Metal effects on the membrane interactions of amyloid-beta peptides.

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.

Liposomal phospholipid preparations of chloramphenicol for ophthalmic applications.

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.