Adam Mechler

Evaporative self-assembly assisted synthesis of polymeric nanoparticles by surface acoustic wave atomization

We demonstrate a straightforward and rapid atomization process driven by surface acoustic waves that is capable of continuously producing spherical monodispersed submicron poly-ε-caprolactone particle aggregates between 150 and 200xa0nm, each of which are composed of nanoparticles of 5-10xa0nm in diameter. The size and morphologies of these particle assemblies were determined using dynamic light scattering, atomic force microscopy and transmission electron microscopy. Through scaling theory, we show that the larger particle aggregates are formed due to capillary instabilities amplified by the acoustic forcing whereas the smaller particulates that form the aggregates arise due to a nucleate templating process as a result of rapid spatially inhomogeneous solvent evaporation. Minimization of the free energy associated with the evaporative process yields a critical cluster size for a single nucleus in the order of 10xa0nm, which roughly corresponds with the dimensions of the sub-50xa0nm particulates.

Transthyretin oligomers induce calcium influx via voltage-gated calcium channels

The deposition of transthyretin (TTR) amyloid in the PNS is a major pathological feature of familial amyloidotic polyneuropathy. The aim of the present study was to examine whether TTR could disrupt cytoplasmic Ca2+ homeostasis and to determine the role of TTR aggregation in this process. The aggregation of amyloidogenic TTR was examined by solution turbidity, dynamic light scattering and atomic force microscopy. A nucleation‐dependent polymerization process was observed in which TTR formed low molecular weight aggregates (oligomers < 100u2003nm in diameter) before the appearance of mature fibrils. TTR rapidly induced an increase in the concentration of intracellular Ca2+ ([Ca2+]i) when applied to SH‐SY5Y human neuroblastoma cells. The greatest effect on [Ca2+]i was induced by a preparation that contained the highest concentration of TTR oligomers. The TTR‐induced increase in [Ca2+]i was due to an influx of extracellular Ca2+, mainly via L‐ and N‐type voltage‐gated calcium channels (VGCCs). These results suggest that increasing [Ca2+]i via VGCCs may be an important early event which contributes to TTR‐induced cytotoxicity, and that TTR oligomers, rather than mature fibrils, may be the major cytotoxic form of TTR.

Synthesis of Ag and Au nanostructures in an ionic liquid: thermodynamic and kinetic effects underlying nanoparticle, cluster and nanowire formation

The in situ reduction of dissolved Ag+ or Au3+ to the zero valent state in the distillable ionic liquid DIMCARB is reported. The reduction process leads to both 1D and 3D nanostructure formation, i.e. nanoparticles and nanowires and clusters composed of nanoparticles. The nanostructures formed have been characterised using UV/vis spectroscopy, powder X-ray diffraction and atomic force microscopic (AFM) topographic and phase imaging. Ag nanostructure growth occurs as a solution based crystallisation process. However, real time AFM imaging of Ag nanostructures formed when poly(vinyl pyrrolidone) is present shows the growth of nanowires on a mica surface via a surface confined process involving Ag atom diffusion. The growth of Au nanowires also occurs via the surface diffusion process. Based on the experimental results, a nanostructure growth mechanism in DIMCARB is proposed and 1D or 3D nanostructure growth is related to either a thermodynamic or a kinetic pathway.

Structure and homogeneity of pseudo-physiological phospholipid bilayers and their deposition characteristics on carboxylic acid terminated self-assembled monolayers.

Supported phospholipid bilayers are frequently used to establish a pseudo-physiological environment required for the study of protein function or the design of enzyme-based biosensors and biocatalytic reactors. These membranes are deposited from bilayer vesicles (liposomes) that rupture and fuse into a planar membrane upon adhesion to a surface. However, the morphology and homogeneity of the resulting layer is affected by the characteristics of the precursor liposome suspension and the substrate. Here we show that two distinct liposome populations contribute to membrane formation--equilibrium liposomes and small unilamellar vesicles. Liposome deposition onto carboxylic acid terminated self-assembled monolayers resulted in planar mono- and multilayer, vesicular and composite membranes, as a function of liposome size and composition. Quartz crystal microbalance data provided estimates for layer thicknesses and sheer moduli and were used for classification of the final structure. Finally, atomic force microscopy data illustrated the inherently inhomogeneous and dynamic nature of these membranes.

Organization of cytochrome P450 enzymes involved in sex steroid synthesis: Protein-protein interactions in lipid membranes

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ± cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.

Cell Penetrating Apidaecin Peptide Interactions with Biomimetic Phospholipid Membranes

Apidaecin peptides from Apis mellifera hemolymph are believed to attack intracellular bacterial targets. Our in vivo results for apidaecins 1a and 1b confirm that bacterial activity is non-lytic, however, the manner in which these peptides pass through the cell membrane to exert this activity is unknown. These data are combined with fluorescence (dye leakage) and quartz crystal microbalance studies to investigate the membrane interaction for these two wildtype peptides. It was found that the peptides penetrate the membrane in a trans-membrane manner. The amount of peptide uptake by the membrane is proportional to the concentration of the peptide, however, this appears to be a dynamic equilibrium which can be almost completely reversed by addition of buffer medium. Interestingly, a small residual mass remains within the membrane and the amount of peptide remaining in the membrane is a function of the buffer-salt concentration viz. in high salt, the residual peptide mass remaining is small whereas at low salt concentration, a larger mass of peptide remains bound. These results support a direct membrane penetration mechanism by the wild type apidaecins 1a and 1b. In both cases the peptide–membrane interaction has a negligible effect on the membrane, although, in high salt a permanent change in the membrane does occur at the highest peptide concentration which does not recover following peptide removal.

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine-FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.

Biochemical and biophysical characterization of a novel plant protein disulfide isomerase

We recently isolated a protein disulfide isomerase (PDI) from the Rubiaceae (coffee family) plant Oldenlandia affinis (OaPDI) and demonstrated that it facilitates the production of disulfide‐knotted defense proteins called cyclotides. PDIs are major folding catalysts in the eukaryotic ER where they are responsible for formation, breakage, or shuffling of disulfide bonds in substrate polypeptides and are important chaperones in the secretory pathway. Here, we report the first detailed analysis of the oligomerization behavior of a plant PDI, based on characterization of OaPDI using various biochemical and biophysical techniques, including size‐exclusion chromatography, NMR spectroscopy, surface plasmon resonance and atomic force microscopy. In solution at low concentration OaPDI comprises mainly monomers, but fractions of dimers and/or higher‐order oligomers were observed at increased conditions, raising the possibility that dimerization and/or oligomerization could be a mechanism to adapt to the various‐sized polypeptide substrates of PDI. Unlike mammalian PDIs, oligomerization of the plant PDI is not driven by the formation of intermolecular disulfide bonds, but by noncovalent interactions. The information derived in this study advances our understanding of the oligomerization behavior of OaPDI in particular but is potentially of broader interest for understanding the mechanism and role of oligomerization, and hence the catalytic and physiological mechanism, of the ubiquitous folding catalyst PDI.

A Study of Protein Electrochemistry on a Supported Membrane Electrode

Protein electrochemistry offers a direct method to identify and characterize biological electron transfer processes, potentially leading to commercial applications such as biosensors and diagnostic tools. However, establishing a biocompatible electrode interface that maintains the native state of the redox protein involves several challenges. In general, membrane proteins require the presence of a phospholipid bilayer to maintain their biological activity. Synthetic `biomimetic’ membranes are widely used to characterize membrane proteins, however they have seldom been applied to measurements of protein redox activity in electrochemical cells due to their inherent insulating property. In this study we demonstrate the use of the phospholipids: PC, PC/PG and PC/PG/cholesterol membrane mixtures on chemically modified (supported) gold electrode surfaces for direct protein electrochemistry. We compare the electrochemical activity of a relatively small, redox active “test protein”, cytochrome c, in the presence and absence of phospholipid on a gold electrode modified with thiol self assembled monolayers, to explore the effect of chain length and composition of the thiol on the charge coupling. Three thiols were investigated as self assembled monolayers on a gold electrode: octanethiol, mercaptopropionic and mercaptoundecanoic acid. We demonstrate here that the charge transfer efficiency of cytochrome c is better in the presence of the membrane and in addition, a superior redox response is obtained with surfaces modified with a thiol functionalised with a carboxylic acid.

Molecular Imaging and Orientational Changes of Antimicrobial Peptides in Membranes

Introduction Resistance to conventional antibiotics has placed antimicrobial peptides under the spotlight as alternative therapeutics for microbial infections. However, the design of specific non-toxic peptides has been elusive due largely to our poor understanding of the precise mechanism of cell lysis. Specifically, the difficulty in defining peptide conformation and location in membranes needs to be addressed. We have used a combination of dual polarisation interferometry (DPI), surface plasmon resonance spectroscopy (SPR) and atomic force microscopy (AFM) to gain an unprecedented detailed molecular picture of membrane lysis by antimicrobial peptides.