Stephen J. Roser

Spectroscopic and X-ray diffraction study of Langmuir–Blodgett films of some 1,4,8,11,15,18-hexaalkyl-22,25-bis(carboxypropyl)phthalocyanines

Molecular assemblies within Langmuir–Blodgett (LB) films of 1,4,8,11,15,18-hexaoctyl-22,25-bis(carboxypropyl)phthalocyanine (1a) copper 1,4,8,11,15,18-hexanonyl-22,25-bis(carboxypropyl)phthalocyaninate (1b) and 1,4,8,11,15,18-hexadecyl-22,25-bis(carboxypropyl)phthalocyanine (1c) have been examined using X-ray diffraction, RAIRS and polarised optical spectroscopy. The LB films are constructed as bilayers by Y-type deposition and the resulting assemblies give rise to visible-region absorption spectra closely similar to those of crystallised films of octaalkylphthalocyanines. When heated, the molecular assemblies within the LB films of 1a and 1b reorganise irreversibly to a form in which the bilayer spacing is reduced. The transition from one type of packing to another is sharp for the film of 1a but occurs over a wide temperature range for the film of 1b. Heating the film of 1c to 140 °C modifies the packing but in this case a further modification occurs on cooling.

Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin

Background: Translocation of the CyaA toxin across plasma membrane is still poorly understood. Results: The region 375–485 is involved in membrane destabilization in vitro and required for cell intoxication. Conclusion: The region 375–485 is crucial for membrane insertion and translocation of the catalytic domain of CyaA. Significance: These results provide new insights on the early stages of the cell intoxication process. The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1–384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1–489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375–485 within CyaA totally abrogated the toxins ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1–384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

Floating lipid bilayers deposited on chemically grafted phosphatidylcholine surfaces

Floating supported bilayers (FSBs) are new systems which have emerged over the past few years to produce supported membrane mimics, where the bilayers remain associated with the substrate, but are cushioned from the substrates constraining influence by a large hydration layer. In this paper we describe a new approach to fabricating FSBs using a chemically grafted phospholipid layer as the support for the floating membrane. The grafted lipid layer was produced using a Langmuir-Schaeffer transfer of acryloyl-functionalized lipid onto a pre-prepared substrate, with AIBN-induced cross-polymerization to permanently bind the lipids in place. A bilayer of DSPC was then deposited onto this grafted monolayer using a combination of Langmuir-Blodgett and Langmuir-Schaeffer transfer. The resulting system was characterized by neutron reflection under two water contrasts, and we show that the new system shows a hydrating layer of approximately 17.5 A in the gel phase, which is comparable to previously described FSB systems. We provide evidence that the grafted substrate is reusable after cleaning and suggest that this greatly simplifies the fabrication and characterization of FSBs compared to previous methods.

Interactions and film formation in polyethylenimine–cetyltrimethylammonium bromide aqueous mixtures at low surfactant concentration

The interactions involved in aqueous mixtures of polyethyleneimine (PEI) and cetyltrimethylammonium bromide (CTAB) were studied under dilute conditions. The phase diagram of this polyelectrolyte-surfactant system of similar charge was constructed by determining the CAC and CMC* values at different PEI concentrations, using surface-tension and conductivity measurements, respectively. Formation of thin films at the air-solution interface was detected at concentrations belonging to the interaction region of the phase diagram, using Brewster angle microscopy. These films were formed at low polymer and surfactant concentrations, 0.01% w/v PEI and 0.1 mmol dm CTAB. Results from SAXS determinations indicate that, under these conditions, mesostructure formation occurs exclusively at the surface. The effect of PEI on surfactant micellisation is determined by the polyelectrolyte nature of the polymer. The presence of the polymer in the aggregate significantly affects free micelle formation, even when hydrophobic interactions are mainly determined by the surfactant structure. The films obtained at low surfactant concentration are mesostructured, composed of five layers, each one 49.7 Å thick, as was determined using specular X-ray reflectometry. These results indicate that mesostructured film formation is achievable under more economical and environmentally friendly conditions, suggesting novel routes for surfactant templating in mixed polyelectrolyte systems of similar charge.

Structure and stability of DPPE planar bilayers

Biomembrane mimics in the form of supported planar bilayers allow the application of a wide range of surface and interface analytical techniques. The structure and phase-behavior of single and double bilayers of 1,2-dipalmitoylphosphoethanolamine (DPPE) were investigated by specular neutron reflectivity for their viability as biomembrane mimics. Whilst single bilayer samples were found to exhibit stable gel and fluid structures, double bilayers were found to be intrinsically unstable in the fluid phase as a planar structure. A Bragg peak was observed in the reflectivity data at just above the gel-to-fluid transition temperature, indicating the partial rearrangement of the upper bilayer into a repeat stacked structure. The lower bilayer was structurally stable. The structure and phase-behaviour of a double bilayer containing a ratio of 9 : 1 DPPE/cholesterol was also investigated to assess the stabilising effect of cholesterol on the upper bilayer. The presence of cholesterol completely destabilised the upper bilayer, causing it to detach 7 °C below the gel-to-fluid transition temperature of DPPE. It is possible that the cholesterol increases the overall conical shape of DPPE molecule by residing in the chain region of the lipid.

X-ray reflection studies on the monolayer-mediated growth of mesostructured MCM-41 silica at the air/water interface

X-Ray reflection has been used to study the nucleation and growth of thin films of a silica–surfactant mesophase (MCM-41) at the air/water interface in the presence and absence of an insoluble lipid monolayer of phosphatidylcholine; the rate of self-assembly and structural order of films comprising up to four micellar layers were enhanced under the lipid monolayer.

Evolution of non-ionic surfactant-templated silicate films at the air–liquid interface

Spontaneous growth of non-ionic surfactant-templated thin films at the air–water interface was investigated using three techniques: Brewster angle microscopy (BAM), time-resolved off-specular X-ray reflectivity and grazing incidence X-ray diffraction (GIXD). Experiments were also carried out to study the evolution of micelles in the subphase solution using small-angle neutron scattering (SANS). Films were prepared in acidic conditions using octaethylene glycol mono-n-hexadecyl ether (C16EO8) as the surfactant and tetramethyloxysilane (TMOS) as the silica precursor. Three different TMOS–C16EO8 molar ratios (3.5, 7.1 and 10.8) were studied. Variation of the silica-precursor concentration causes a significant effect on the film-formation time, the solution and film-growth mechanisms and the final film structure.

In situ neutron reflectivity studies of electroactive films

In situ neutron reflectivity measurements on potentiodynamically deposited polybithiophene (PBT) films on Pd electrodes supported on quartz are described. The contrast-variation method allows determination of polymer film structure with high resolution perpendicular to the electrode surface. Both undoped and p-doped PBT films comprised a dense inner region, containing virtually no solvent, and an extended diffuse outer region containing considerable solvent. The p-doped films are slightly more diffuse and thicker than the undoped films. Films held in the p-doped state for an extended period of time retain that structure upon undoping. Neutron reflectivity and coulometric data suggest that p-doped films (at +1.2 V) carry one positive charge for every three thiophene rings.

In situ neutron reflectivity studies of polybithiophene

In situ neutron reflection experiments have been used to obtain the thickness and density profile of polybithiophene films perpendicular to the surface of a Pd electrode. Undoped films are dense and contain little solvent or electrolyte. The scattering length density of the neutral (undoped) polymer allows estimation of the amount of polymer and hence the charge density per thiophene ring on the oxidised (doped) form of the polymer.

Spontaneous free-standing nanostructured film growth in polyelectrolyte-surfactant systems

Substitution of a polyelectrolyte for silica during formation of surfactant-templated films produces similar nano- and macroscale structures confirming that silica acts as a polyelectrolyte during thin film self-assembly.