Assembling the Tat protein translocase.

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

Micron dimensioned cavity array supported lipid bilayers for the electrochemical investigation of ionophore activity.

Microcavity supported lipid bilayers, MSLBs, were applied to an electrochemical investigation of ionophore mediated ion transport. The arrays comprise of a 1cm(2) gold electrode imprinted with an ordered array of uniform spherical-cap pores of 2.8µm diameter prepared by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aqueous buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by the micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coefficient of 12.58±1.28µm(2)s(-1) and anomalous exponent of 1.03±0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3days and electrochemical impedance spectroscopy of the membrane with and without ionic gradient over experimental windows of 6h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K(+) uniporter and Nigericin, a K(+)/H(+) antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance respectively. Significant increases in Valinomycin and Nigericin activity were observed, reflected in large decreases in membrane resistance when K(+) was present in the contacting buffer and in the presence of H(+) ionic gradient across the membrane respectively.

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild-type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.

Aqueous-filled polymer microcavity arrays: versatile & stable lipid bilayer platforms offering high lateral mobility to incorporated membrane proteins.

A key prerequisite in an ideal supported lipid bilayer based cell membrane model is that the mobility of both the lipid matrix and its components are unhindered by the underlying support. This is not trivial and with the exception of liposomes, many of even the most advanced approaches, although accomplishing lipid mobility, fail to achieve complete mobility of incorporated membrane proteins. This is addressed in a novel platform comprising lipid bilayers assembled over buffer-filled, arrays of spherical cap microcavities formed from microsphere template polydimethoxysilane. Prior to bilayer assembly the PDMS is rendered hydrophilic by plasma treatment and the lipid bilayer prepared using Langmuir Blodgett assembly followed by liposome/proteoliposome fusion. Fluorescence Lifetime Correlation Spectroscopy confirmed the pore suspended lipid bilayer exhibits diffusion coefficients comparable to free-standing vesicles in solution. The bilayer modified arrays are highly reproducible and stable over days. As the bilayers are suspended over deep aqueous reservoirs, reconstituted membrane proteins experience an aqueous interface at both membrane interfaces and attain full lateral mobility. Their utility as membrane protein platforms was exemplified in two case studies with proteins of different dimensions in their extracellular and cytoplasmic domains reconstituted into DOPC lipid bilayers; Glycophorin A, and Integrin αIIbß3. In both cases, the proteins exhibited 100% mobility with high lateral diffusion coefficients.

Fluorescence correlation and lifetime correlation spectroscopy applied to the study of supported lipid bilayer models of the cell membrane.

Supported Lipid Bilayers (SLBs) are versatile models capable of mimicking some of the key properties of the cell membrane, including for example lipid fluidity, domain formation and protein support, without the challenging complexity of the real biological system. This is important both from the perspective of understanding the behaviour and role of the lipid membrane in cell structure and signalling, as well as in development of applications of lipid membranes across domains as diverse as sensing and drug delivery. Lipid and protein diffusion within the membrane is vital to its function and there are several key experimental methods used to study membrane dynamics. Amongst the optical methods are Fluorescence Recovery After Photobleaching (FRAP), single particle tracking and Fluorescence Correlation (and Fluorescence Lifetime Correlation) Spectroscopy (FCS/FLCS). Each of these methods can provide different and often complementary perspectives on the dynamics of the fluid membrane. Although FCS is well established, FLCS is a relatively new technique and both methods have undergone a number of extensions in recent years which improve their precision and accuracy in studying supported lipid bilayers, most notably z-scan methods. This short review focusses on FCS and FLCS and their recent applications, specifically to artificial lipid bilayer studies addressing key issues of cell membrane behaviour.

Multilayer assemblies of polyelectrolyte-gold nanoparticles for the electrocatalytic oxidation and detection of arsenic(III).

Multilayers of poly(diallyldimethylammonium chloride) (PDDA) and citrate capped Au nanoparticles (AuNPs) anchored on sodium 3-mercapto-1-propanesulfonate modified gold electrode by electrostatic layer-by-layer assembly (LbL) technique are shown to be an excellent architecture for the direct electrochemical oxidation of As(III) species. The growth of successive layers in the proposed LbL architecture is followed by atomic force microscopy, UV-vis spectroscopy, quartz crystal microbalance with energy dissipation, and electrochemistry. The first bilayer is found to show rather different physico-chemical characteristics as compared to the subsequent bilayers, and this is attributed to the difference in the adsorption environments. The analytical utility of the architecture with five bilayers is exploited for arsenic sensing via the direct electrocatalytic oxidation of As(III), and the detection limit is found to be well below the WHO guidelines of 10ppb. When the non-redox active PDDA is replaced by the redox-active Os(2,2'-bipyridine)(2)Cl-poly(4-vinylpyridine) polyelectrolyte (PVPOs) in the LbL assembly, the performance is found to be inferior, demonstrating that the redox activity of the polyelectrolyte is futile as far as the direct electro-oxidation of As(III) is concerned.

Amphipol mediated surface immobilization of FhuA: a platform for label-free detection of the bacteriophage protein pb5.

Biotinylated amphipol was used to entrap FhuA (an E. coli outer membrane protein) and immobilize the FhuA-amphipol complex on streptavidin surfaces. Using this assembly, we have successfully devised surface-based assays for studying the recognition of FhuA by pb5 (a bacteriophage T5 protein) and determination of the affinity constant.

Tethered bilayer lipid membranes on mixed self-assembled monolayers of a novel anchoring thiol: impact of the anchoring thiol density on bilayer formation.

Tethered bilayer lipid membranes (tBLMs) are designed on mixed self-assembled monolayers (SAMs) of a novel synthetic anchoring thiol, 2,3-di-o-palmitoylglycerol-1-tetraethylene glycol mercaptopropanoic acid ester (TEG-DP), and a new short dilution thiol molecule, tetraethylene glycol mercaptopropanoic acid ester (TEG). tBLM formation was accomplished by self-directed fusion of small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The influence of the dilution of the anchoring thiol molecule in the SAM on the vesicle fusion process and on the properties of the resulting tBLMs is studied. It is observed by quartz crystal microbalance that vesicle fusion is a one-step process for a pure TEG-DP SAM as well as for mixed SAMs containing a high concentration of the anchoring thiol. However, upon dilution of the anchoring thiol to moderate concentrations, this process is decelerated and possibly follows a pathway different from that observed on a pure TEG-DP SAM. Electrochemical impedance spectroscopy is used to qualitatively correlate the composition of the SAM to the electrical properties of the tBLM. In this paper we also delineate the necessity of a critical concentration of this anchoring TEG-DP thiol as a requisite for inducing the fusion of vesicles to form a tBLM.

Cell adhesion through clustered ligand on fluid supported lipid bilayers.

The quartz crystal microbalance technique with dissipation monitoring and the complementary optical microscopy technique were used for monitoring cell adhesion on an RGD-functionalized supported lipid bilayer. A critical interligand RGD spacing of nearly 80 nm was estimated for cell adhesion when an RGD spacing of 10 nm was necessary to observe flattened cells on a fluid surface.

Two-component hydrogels comprising fatty acids and amines: structure, properties, and application as a template for the synthesis of metal nanoparticles.

Stearic acid or eicosanoic acid mixed with di- or oligomeric amines in specific molar ratios form stable gels in water. The formation of such hydrogels depends on the hydrophobicity of the fatty acid, and also on the type of amine used. The gelation properties of these two-component systems were investigated using electron microscopy, FTIR spectroscopy, 1H NMR spectroscopy, differential scanning calorimetry (DSC), and both single-crystal and cast-film X-ray diffraction. Results of FTIR spectral analysis suggest salt formation during gelation. 1H NMR analysis of the gels indicates that the fatty acid chains are immobilized in the gel state and when the gel melts, these chains regain their mobility. Analysis of DSC data indicates that increase in the spacer length in the di-/oligomeric amine lowers the gel-melting temperature. Two of these gelator salts developed into crystals and structural details of such systems could be secured by single-crystal X-ray diffraction analysis. The structural information of the salts thus obtained was compared with the XRD data of the self-supporting films of those gels. Such analyses provided pertinent structural insight into the supramolecular interactions that prevail within these gelator assemblies. Analysis of the crystal structure confirmed that multilayered lamellar aggregates exist in the gel and it also showed that the three-dimensional ordering observed in the crystalline phase is retained in only one direction in the gel state. Finally, the hydrogel was used as a medium for the synthesis of silver nanoparticles. The nanoparticles were found to position themselves on the fibers and produced a long, ordered assembly of gel-nanoparticle composite.