How Membrane Geometry Regulates Protein Sorting Independently of Mean Curvature.

Biological membranes have distinct geometries that confer specific functions. However, the molecular mechanisms underlying the phenomenological geometry/function correlations remain elusive. We studied the effect of membrane geometry on the localization of membrane-bound proteins. Quantitative comparative experiments between the two most abundant cellular membrane geometries, spherical and cylindrical, revealed that geometry regulates the spatial segregation of proteins. The measured geometry-driven segregation reached 50-fold for membranes of the same mean curvature, demonstrating a crucial and hitherto unaccounted contribution by Gaussian curvature. Molecular-field theory calculations elucidated the underlying physical and molecular mechanisms. Our results reveal that distinct membrane geometries have specific physicochemical properties and thus establish a ubiquitous mechanistic foundation for unravelling the conserved correlations between biological function and membrane polymorphism.

Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases.

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane curvature was essential for enrichment in raft-like liquid-ordered phases; enrichment was driven by relief of lateral pressure upon anchor insertion and most likely affects the localization of lipidated proteins in general.

Triazatriangulene as binding group for molecular electronics.

The triazatriangulene (TATA) ring system was investigated as a binding group for tunnel junctions of molecular wires on gold surfaces. Self-assembled monolayers (SAMs) of TATA platforms with three different lengths of phenylene wires were fabricated, and their electrical conductance was recorded by both conducting probe-atomic force microscopy (CP-AFM) and scanning tunneling microscopy (STM). Similar measurements were performed for phenylene SAMs with thiol anchoring groups as references. It was found that, despite the presence of a sp(3) hybridized carbon atom in the conduction path, the TATA platform displays a contact resistance only slightly larger than the thiols. This surprising finding has not been reported before and was analyzed by theoretical computations of the transmission functions of the TATA anchored molecular wires. The relatively low contact resistance of the TATA platform along with its high stability and directionality make this binding group very attractive for molecular electronic measurements and devices.

Wet chemical synthesis of soluble gold nanogaps.

A central challenge in molecular electronics is to create electrode pairs separated by only a few nanometers that can accommodate a single molecule of interest to be optically or electrically characterized while residing in the gap. Current techniques for nanogap fabrication are largely based on top-down approaches and often rely on subsequent deposition of molecules into the nanogap. In such an approach, the molecule may bridge the gap differently with each experiment due to variations at the metal-molecule interface. Conversely, chemists can readily synthesize gold nanorods (AuNRs) in aqueous solution. Through controlled end-to-end assembly of the AuNRs into dimers or chains, facilitated via target molecules, they can be used as electrical contacts. In this way, the preparation of AuNR-molecule-AuNR junctions by wet chemical methods may afford a large number of identical devices with little variation in the interface between molecule and electrode (AuNR). In this Account, we highlight recent progress in using chemically synthesized AuNRs as building blocks for molecular electronic applications. We outline the general synthesis and properties of AuNRs and describe the aqueous growth of dimeric AuNR structures from an insulating molecule linked to AuNR precursors (gold seeds). Conjugated, electronically active molecules are typically not soluble under the conditions required for the bottom-up growth of AuNRs. Therefore, we present a strategy that utilizes host-guest chemistry in order to make such π-systems compatible with the AuNR growth procedure. In order to electrically characterize the AuNR-molecule-AuNR constructs, we must transfer them onto a substrate and contact external electrodes. We discuss the implications of using electron-beam lithography for making this contact. In addition, we introduce a novel fabrication approach in which we can grow AuNR nanogap electrodes in situ on prepatterned substrates, thus circumventing post-processing steps that potentially damage the nanogap environment. Due to the inherent optical properties of AuNRs, electromagnetic field enhancement in the nanogaps lets us spectroscopically characterize the molecules via surface-enhanced Raman scattering. We discuss the incorporation of oligopeptides functionalized with acetylene units having uniquely identifiable vibrational modes. This acetylene moiety allows chemical reactions to be performed in the gaps via click chemistry, and the oligopeptide linking platform opens for integration of larger biological components.

Deterministic assembly of linear gold nanorod chains as a platform for nanoscale applications.

We demonstrate a method to assemble gold nanorods highly deterministically into a chain formation by means of directed capillary assembly. This way we achieved straight chains consisting of end-to-end aligned gold nanorods assembled in one specific direction with well-controlled gaps of ∼6 nm between the individual constituents. We determined the conditions for optimum quality and yield of nanorod chain assembly by investigating the influence of template dimensions and assembly temperature. In addition, we transferred the gold nanorod chains from the assembly template onto a Si/SiO2 target substrate, thus establishing a platform for a variety of nanoscale electronic and optical applications ranging from molecular electronics to optical and plasmonic devices. As a first example, electrical measurements are performed on contacted gold nanorod chains before and after their immersion in a solution of thiol end-capped oligophenylenevinylene molecules showing an increase in the conductance by three orders of magnitude, indicating molecular-mediated transport.

Ultrathin reduced graphene oxide films as transparent top-contacts for light switchable solid-state molecular junctions.

A new type of solid-state molecular junction is introduced, which employs reduced graphene oxide as a transparent top contact that permits a self-assembled molecular monolayer to be photoswitched in situ, while simultaneously enabling charge-transport measurements across the molecules. The electrical switching behavior of a less-studied molecular switch, dihydroazulene/vinylheptafulvene, is described, which is used as a test case.

Quantum interference effects at room temperature in OPV-based single-molecule junctions.

Interference effects on charge transport through an individual molecule can lead to a notable modulation and suppression on its conductance. In this letter, we report the observation of quantum interference effects occurring at room temperature in single-molecule junctions based on oligo(3)-phenylenevinylene (OPV3) derivatives, in which the central benzene ring is coupled to either para- or meta-positions. Using the break-junction technique, we find that the conductance for a single meta-OPV3 molecule wired between gold electrodes is one order of magnitude smaller than that of a para-OPV3 molecule. Theoretical calculations confirm the occurrence of constructive and destructive interference in the para- and meta-OPV3 molecules respectively, which arises from the phase difference of the transmission coefficients through the molecular orbitals.

Bonding and electronic transport properties of fullerene and fullerene derivatives in break-junction geometries.

Fullerenes are considered anchoring groups for molecular electronics due to a large contact area and their affinity for noble metals. The conductances of fullerene-terminated molecules, however, are found to be even lower than for thiol termination. The effects of weak molecule-metal coupling and symmetry breaking are studied by transport measurements of C(60) and functionalized C(60). The results demonstrate highy efficient contacts between Au and C(60), despite of deposition from solution.

Electrical annealing and temperature dependent transversal conduction in multilayer reduced graphene oxide films for solid-state molecular devices.

The transversal conductance through thin multi-layered films of reduced graphene oxide was studied as a function of temperature in a solid-state device setup designed for molecular electronic measurements. Upon cooling to cryogenic temperatures, the resistivity of the films increased by about three orders of magnitude compared to the value at room temperature, and this temperature dependence was described by a variable range hopping model. Above a certain threshold voltage the films could be annealed electrically at low temperatures. This electrical annealing resulted in a dramatic decrease in resistivity by up to four orders of magnitude. Upon reheating, the conductivity of the annealed films displayed an almost negligible temperature dependence. These results are promising for the application of reduced graphene oxide as a soft top-contact layer for molecular monolayer devices in the solid-state.

A triptycene-based approach to solubilising carbon nanotubes and C60.

We describe herein the synthesis of a triptycene-based surfactant designed with the ability to solubilise single-walled carbon nanotubes (SWNTs) and C(60) in water through non-covalent interactions. Furthermore, an amphiphilic naphthalene-based surfactant with the same ability to solubilise SWNTs and C(60) has also been prepared. The compounds synthesised were designed with either two ionic or non-ionic tails to ensure a large number of supramolecular interactions with the solvent, thereby promoting strong solubilisation. The surfactants produced stable suspensions in which the SWNTs are dispersed and the surfactant/SWNT complexes formed are stable for more than one year. UV/Vis/NIR absorption spectroscopy, TEM and AFM were employed to probe the solubilisation properties of the dispersion of surfactants and SWNTs in water.

End-to-end assembly of gold nanorods via oligopeptide linking and surfactant control.

We report two novel approaches for fabricating self-assembled chains of end-to-end linked Au nanorods separated by a nanogap. In one approach, bi-functional cysteine end-capped oligopeptides of different lengths are used as the linking agent. The widths of the produced nanogaps scale with the length and tertiary structure of the peptide linker. Functionalized oligopeptides containing an acetylene group are also employed as a linker, and the functional group is uniquely identified using surface-enhanced Raman spectroscopy. The development of an oligopeptide-linking platform is motivated by the ease of synthesis and high modularity of peptides; these features enable the possibility to integrate diverse functionality into molecular nanogap junctions - synthesized in water. The stepwise nanochain formation is followed via the evolution of the longitudinal plasmon absorption band in combination with transmission electron microscopy. The reaction rate and extent is tuned by controlling the concentration of the stabilizing CTAB surfactant in the solution. At very low surfactant concentrations, spontaneous end-to-end linking of the Au nanorods is observed even in the absence of linking peptide. The assembled AuNRs may act as next-generation electrodes in a platform for molecular electronics and synthetic biology.

Aligned growth of gold nanorods in PMMA channels: parallel preparation of nanogaps.

We demonstrate alignment and positional control of gold nanorods grown in situ on substrates using a seed-mediated synthetic approach. Alignment control is obtained by directing the growth of spherical nanoparticle seeds into nanorods in well-defined poly(methyl methacrylate) nanochannels. Substrates with prepatterned metallic electrodes provide an additional handle for the position of the gold nanorods and yield nanometer-sized gaps between the electrode and nanorod. The presented approach is a novel demonstration of bottom-up device fabrication of multiple nanogap junctions on a single chip mediated viain situ growth of gold nanorods acting as nanoelectrodes.

Solution-processed ultrathin chemically derived graphene films as soft top contacts for solid-state molecular electronic junctions.

A novel method using solution-processed ultrathin chemically derived graphene films as soft top contacts for the non-destructive fabrication of molecular junctions is demonstrated. We believe this protocol will greatly enrich the solid-state test beds for molecular electronics due to its low-cost, easy-processing and flexible nature.

Molecular junctions based on SAMs of cruciform oligo(phenylene ethynylene)s.

Cruciform oligo(phenylene ethynylene)s (OPEs) with an extended tetrathiafulvalene (TTF) donor moiety (OPE5-TTF and OPE3-TTF) and their simple analogues (OPE5-S and OPE3) without conjugated substituents were used to form high-quality self-assembled monolayers (SAMs) on ultraflat gold substrates. Molecular junctions based on these SAMs were investigated using conducting-probe atomic force microscopy (CP-AFM). The TTF substituent changes the molecular orbital energy levels and decreases the HOMO-LUMO energy gap, resulting in a 9-fold increase in conductance for both TTF cruciform OPEs compared to the unsubstituted analogues. The difference in electrical transport properties of the SAMs was reproduced by the theoretical transport calculations for the single molecules.

Influence of the preparation route on the supramolecular organization of lipids in a vesicular system.

A confocal fluorescence microscopy-based assay was used for studying the influence of the preparation route on the supramolecular organization of lipids in a vesicular system. In this work, vesicles composed of cholesterol and CTAB (1/1 mol %) or cholesterol and DOPC (2/8 mol %) and incorporating two membrane dyes were prepared by either a compressed fluid (CF)-based method (DELOS-susp) or a conventional film hydration procedure. They were subsequently immobilized and imaged individually using a confocal fluorescence microscope. Two integrated fluorescence intensities, I(dye1) and I(dye2), were assigned to each tracked vesicle, and their ratio, I(dye1)/I(dye2), was used for quantifying the degree of membrane inhomogeneity between individual vesicles within each sample. A distribution of I(dye1)/I(dye2) values was obtained for all the studied vesicular systems, indicating intrasample heterogeneity. The degree of inhomogeneity (DI) was similar for Chol/DOPC vesicles prepared by both procedures. In contrast, DI was more than double for the hydration method compared to the CF-based method in the case of Chol/CTAB vesicles, which can suffer from lipid demixing during film formation. These findings reveal a more homogeneous vesicle formation path by CFs, which warranted good homogeneity of the vesicular system, independently of the lipid mixture used.

Aligning nanodiscs at the air-water interface, a neutron reflectivity study.

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 Å with a surface coverage of 66 ± 4%. This layer is located about 15 Å below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 Å) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.

Savinase action on bovine serum albumin (BSA) monolayers demonstrated with measurements at the air-water interface and liquid Atomic Force Microscopy (AFM) imaging.

We studied the enzymatic action of Savinase on bovine serum albumin (BSA) organized in a monolayer spread at the air/water interface or adsorbed at the mica surface. We carried out two types of experiments. In the first one we followed the degradation of the protein monolayer by measuring the surface pressure and surface area decrease versus time. In the second approach we applied AFM imaging of the supported BSA monolayers adsorbed on mica solid supports and extracted information for the enzyme action by analyzing the obtained images of the surface topography in the course of enzyme action. In both cases we obtained an estimate for the turnover number (TON) of the enzyme reaction.

Voltammetry and in situ scanning tunnelling spectroscopy of osmium, iron, and ruthenium complexes of 2,2':6',2''-terpyridine covalently linked to Au(111)-electrodes.

We have studied self-assembled molecular monolayers (SAMs) of complexes between Os(II)/(III), Fe(II)/(III), and Ru(II)/(III) and a 2,2',6',2''-terpyridine (terpy) derivative linked to Au(111)-electrode surfaces via a 6-acetylthiohexyloxy substituent at the 4'-position of terpy. The complexes were prepared in situ by first linking the terpy ligand to the surface via the S-atom, followed by addition of suitable metal compounds. The metal-terpy SAMs were studied by cyclic voltammetry (CV), and in situ scanning tunnelling microscopy with full electrochemical potential control of substrate and tip (in situ STM). Sharp CV peaks were observed for the Os- and Fe complexes, with interfacial electrochemical electron transfer rate constants of 6-50 s(-1). Well-defined but significantly broader peaks (up to 300 mV) were observed for the Ru-complex. Addition of 2,2'-bipyridine (bipy) towards completion of the metal coordination spheres induced voltammetric sharpening. In situ STM images of single molecular scale strong structural features were observed for the osmium and iron complexes. As expected from the voltammetric patterns, the surface coverage was by far the highest for the Ru-complex which was therefore selected for scanning tunnelling spectroscopy. These correlations displayed a strong peak around the equilibrium potential with systematic shifts with increasing bias voltage, as expected for a sequential two-step in situ ET mechanism.

A statistical approach to inelastic electron tunneling spectroscopy on fullerene-terminated molecules.

We report on the vibrational fingerprint of single C(60) terminated molecules in a mechanically controlled break junction (MCBJ) setup using a novel statistical approach manipulating the junction mechanically to address different molecular configurations and to monitor the corresponding vibrational modes. In the IETS spectra, the vibrations of the anchoring C(60) dominate the spectra; thus information on the unit anchored with C(60) to the electrodes is masked by the modes arising from the anchoring groups. However, we have identified the additional modes from the fluorene backbone optically.