A Decaheme Cytochrome as a Molecular Electron Conduit in Dye-Sensitized Photoanodes.

In nature, charge recombination in light-harvesting reaction centers is minimized by efficient charge separation. Here, it is aimed to mimic this by coupling dye-sensitized TiO2 nanocrystals to a decaheme protein, MtrC from Shewanella oneidensis MR-1, where the 10 hemes of MtrC form a ≈7-nm-long molecular wire between the TiO2 and the underlying electrode. The system is assembled by forming a densely packed MtrC film on an ultra-flat gold electrode, followed by the adsorption of approximately 7 nm TiO2 nanocrystals that are modified with a phosphonated bipyridine Ru(II) dye (RuP). The step-by-step construction of the MtrC/TiO2 system is monitored with (photo)electrochemistry, quartz-crystal microbalance with dissipation (QCM-D), and atomic force microscopy (AFM). Photocurrents are dependent on the redox state of the MtrC, confirming that electrons are transferred from the TiO2 nanocrystals to the surface via the MtrC conduit. In other words, in these TiO2/MtrC hybrid photodiodes, MtrC traps the conduction-band electrons from TiO2 before transferring them to the electrode, creating a photobioelectrochemical system in which a redox protein is used to mimic the efficient charge separation found in biological photosystems.

The morphology of decorated amyloid fibers is controlled by the conformation and position of the displayed protein.

Self-assembled structures capable of mediating electron transfer are an attractive scientific and technological goal. Therefore, systematic variants of SH3-Cytochrome b(562) fusion proteins were designed to make amyloid fibers displaying heme-b(562) electron transfer complexes. TEM and AFM data show that fiber morphology responds systematically to placement of b(562) within the fusion proteins. UV-vis spectroscopy shows that, for the fusion proteins under test, only half the fiber-borne b(562) binds heme with high affinity. Cofactor binding also improves the AFM imaging properties and changes the fiber morphology through changes in cytochrome conformation. Systematic observations and measurements of fiber geometry suggest that longitudinal registry of subfilaments within the fiber, mediated by the interaction and conformation of the displayed proteins and their interaction with surfaces, gives rise to the observed morphologies, including defects and kinks. Of most interest is the role of small molecule modulation of fiber structure and mechanical stability. A minimum complexity model is proposed to capture and explain the fiber morphology in the light of these results. Understanding the complex interplay between these factors will enable a fiber design that supports longitudinal electron transfer.

Differing modes of interaction between monomeric Aß(1-40) peptides and model lipid membranes: an AFM study.

Membrane interactions with ß-amyloid peptides are implicated in the pathology of Alzheimer's disease and cholesterol has been shown to be key modulator of this interaction, yet little is known about the mechanism of this interaction. Using atomic force microscopy, we investigated the interaction of monomeric Aß(1-40) peptides with planar mica-supported bilayers composed of DOPC and DPPC containing varying concentrations of cholesterol. We show that below the bilayer melting temperature, Aß monomers adsorb to, and assemble on, the surface of DPPC bilayers to form layers that grow laterally and normal to the bilayer plane. Above the bilayer melting temperature, we observe protofibril formation. In contrast, in DOPC bilayers, Aß monomers exhibit a detergent-like action, forming defects in the bilayer structure. The kinetics of both modes of interaction significantly increases with increasing membrane cholesterol content. We conclude that the mode and rate of the interaction of Aß monomers with lipid bilayers are strongly dependent on lipid composition, phase state and cholesterol content.

Crystalline hydration structure at the membrane-fluid interface of model lipid rafts indicates a highly reactive boundary region.

The fluid mosaic model of biological membranes is that of a two-dimensional lipid bilayer in which both lipids and associated membrane proteins diffuse freely. More recently, the raft hypothesis proposed that membranes contain small, dynamic, functional domains (rafts), which act as platforms for membrane protein attachment and interaction. Although experimental evidence supporting the raft hypothesis is growing, very little is known of the structure of the membrane-fluid interface of lipid raft systems. Here, we report the direct submolecular-scale imaging of model raft membranes using ultrahigh resolution atomic force microscopy. We characterize the heterogeneous nature of crystalline hydration layers at the membrane-fluid interface. The association of crystalline hydration layers with raft membranes would significantly affect the mechanism and kinetics of both inter-raft interactions and those between rafts and external biomolecules, and therefore this finding has important implications for membrane biology.

Direct submolecular scale imaging of mesoscale molecular order in supported dipalmitoylphosphatidylcholine bilayers.

Supported dipalmitoylphosphatidylcholine (DPPC) bilayers are widely used membrane systems in biophysical and biochemical studies. Previously, short-range positional and orientational order of lipid headgroups of supported DPPC bilayers was observed at room temperature using low deflection noise frequency modulation atomic force microscopy (FM-AFM). While this ordering was supported by X-ray diffraction studies, it conflicted with diffusion coefficient measurements of gel-phase bilayers determined from fluorescence photobleaching experiments. In this work, we have directly imaged mica-supported DPPC bilayers with submolecular resolution over scan ranges up to 146 nm using low deflection noise FM-AFM. Both orientational and positional molecular ordering were observed in the mesoscale, indicative of crystalline order. We discuss these results in relation to previous biophysical studies and propose that the mica support induces mesoscopic crystalline order of the DPPC bilayer at room temperature. This study also demonstrates the recent advance in the scan range of submolecular scale AFM imaging.

Nanoscale mechanical probing of supported lipid bilayers with atomic force microscopy.

We present theory and experiments for the force-distance curve F(z(0)) of an atomic force microscope (AFM) tip (radius R) indenting a supported fluid bilayer (thickness 2d). For realistic conditions the force is dominated by the area compressibility modulus κ(A) of the bilayer and, to an excellent approximation, given by F=πκ(A)Rz(0)(2)/(2d-z(0))(2). The experimental AFM force curves from coexisting liquid ordered and liquid disordered domains in three-component lipid bilayers are well described by our model, which provides κ(A) in agreement with literature values. The liquid ordered phase has a yieldlike response that we model as due to the breaking of hydrogen bonds.

Manipulation and charge determination of proteins in photopatterned solid supported bilayers.

This work demonstrates the use of deep UV micropatterned chlorotrimethylsilane (TMS) monolayers to support lipid membranes on SiO(2) surfaces. After immersing such a patterned surface into a solution containing small unilamellar vesicles of egg PC, supported bilayer lipid membranes were formed on the hydrophilic, photolyzed regions and lipid monolayer over the hydrophobic, non-photolyzed regions. A barrier between the lipid monolayer and bilayer regions served to stop charged lipids migrating between the two. This allows the system to be used to separate charged lipids or proteins by electrophoresis. Either oppositely charged fluorescence labeled lipids [Texas Red DHPE (negative charge) and D291 (positive charge)] or lipids with different charge numbers [Texas Red DHPE (one negative charge) and NBD PS (two negative charges)] can be separated. We have also studied the migration of streptavidin attached to a biotinylated lipid. Negatively charged streptavidin responds to the applied electric field by moving in the direction of electroosmotic flow, i.e. towards the negative electrode. However the direction of streptavidin movement can be controlled by altering the difference in zeta potential between that of the streptavidin (zeta(1)) and the lipid membrane (zeta(2)). If zeta(1) > zeta(2), streptavidin moves to the negative electrode, while if zeta(1) < zeta(2), streptavidin moves to the positive electrode. This balance was manipulated by adding positively charged lipid DOTAP to the membrane. After measuring the average drift velocity of streptavidin as a function of DOTAP concentration, the point where zeta(1) approximately zeta(2) was found. At this point zeta(1) was calculated to be -9.8 mV which is in good agreement with the value of -13 mV from force measurements and corresponds to a charge of -2e per streptavidin, thus demonstrating the applicability of this method for determining protein charge.

Minimal F-actin cytoskeletal system for planar supported phospholipid bilayers.

Preferential binding of F-actin to lipid bilayers containing ponticulin was investigated on both planar supported bilayers and on a cholesterol-based tethering system. The transmembrane protein ponticulin in Dictyostelium discoideum is known to provide a direct link between the actin cytoskeleton and the cell membrane ( Wuestehube, L. J. ; Luna, E. J. J. Cell Biol. 1987, 105, 1741- 1751 ). Purification of ponticulin has allowed an in vitro model of the F-actin cytoskeletal scaffold system to be formed and investigated by AFM, epi-fluorescence microscopy, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D). Single filament features of F-actin bound to the ponticulin containing lipid bilayer are shown by AFM to have a pitch of 37.3 +/- 1.1 nm and a filament height of 7.0 +/- 1.6 nm. The complementary techniques of QCM-D and SPR were used to obtain dissociation constants for the interaction of F-actin with ponticulin containing bilayers, giving 10.5 +/- 1.7 microM for a physisorbed bilayer and 10.8 +/- 3.6 microM for a tethered bilayer, respectively.

Supported bilayer lipid membrane arrays on photopatterned self-assembled monolayers.

This work demonstrates the use of photocleavable cholesterol derivatives to create supported bilayer lipid membrane arrays on silica. The photocleavable cholesteryl tether is attached to the surface by using the reaction of an amine-functionalized self-assembled monolayer (SAM) and the N-hydroxysuccinimide-based reagent 9. The resultant SAM contains an ortho-nitrobenzyl residue that can be cleaved by photolysis by using soft (365 nm) UV light regenerating the original amine surface, and which can be patterned using a mask. The photoreaction yield was approximately 75 % which was significantly higher than previously found for related ortho-nitrobenzyl photochemistry on gold substrates. The SAMs were characterized by means of contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Patterned surfaces were characterized with SEM and AFM. After immersing the patterned surface into a solution containing small unilamellar vesicles of egg phosphatidylcholine (PC), supported lipid membranes were formed comprised of lipid bilayer over the amine functionalized "hydrophilic" regions and lipid monolayer over the cholesteryl "hydrophobic" regions. This was confirmed by fluorescence microscopy and AFM. FRAP studies yielded a lateral diffusion coefficient for the probe molecule of 0.14+/-0.05 microm(2) s(-1) in the bilayer regions and approximately 0.01 microm(2) s(-1) in the monolayer regions. This order of magnitude difference in diffusion coefficients effectively serves to isolate the bilayer regions from one another, thus creating a bilayer array.

Titration of ionizable monolayers by measurement of the electric double-layer force.

We have determined the pKa of surface-bound primary amine groups by determining the surface potential as a function of solution pH from the magnitude of the electric double-layer force. Using colloid-probe atomic force microscopy (AFM), we measured the force as a function of separation between a particle of radius R = 10 microm and a planar surface, each coated with a self-assembled monolayer of HS(CH2)2CONH((CH2)2O)8(CH2)2NH2. The force was measured from pH 3 to 7, and the surface potential was determined by fitting the results to solutions of the nonlinear Poisson-Boltzmann equation. The surface pKa of the primary amine group was found to be 5.0 +/- 0.2, in agreement with the results of contact-angle and chemical-force titrations on similar surfaces with primary amine groups. The surface charge density indicates that less than 1% of the NH2 groups are dissociated at pH 3, suggesting that ionization is very unfavorable in the local environment of the ethylene oxide chains. This noncontact method should be of general applicability to surfaces with ionizable groups and avoids the possible complications of large contact forces on the surfaces under study.

A model system to study the insertion of cholesterol into a phospholipid monolayer.

Colloidal probe atomic force microscopy (AFM) was used to study the interaction between a surface bearing tethered cholesterol groups and an egg phosphatidylcholine (egg-PC) monolayer. The cholesterol bearing surface was comprised of a mixed self-assembled monolayer comprised of O-cholesteryl N-(8'-mecapto-3',6'-dioxaoctyl)carbamate (CPEO3) molecules and beta-mercaptoethanol formed on a 20 mum diameter gold-coated silica particle. The egg-PC monolayer was adsorbed onto an octadecylthiol monolayer formed on template-stripped gold. The force between the surfaces, as a function of separation, was measured for surface concentrations of CPEO3 from 0 to 100 mol %. At all concentrations there was a long-range repulsive double-layer force due to weak surface charges. At surface concentrations of CPEO3 from 1 to 29 mol % the interaction on the approach of the surfaces showed a maximum in the repulsive force, followed by a small (2-5 nm) jump into a force minimum corresponding to adhesion of the surfaces. On separation, a normalized pull-off force of 1.0-1.6 mN m(-1) was measured. Over the same concentration range, the calculated interaction energy per CPEO3 molecule decreased from 1.1 +/- 0.2 kT to 0.04 kT. At surface concentrations of 35 mol % and above there was no reproducible adhesion between the cholesterol-bearing surface and the phospholipid monolayer. We attribute the occurrence of short-range attraction and adhesion in the 1-29 mol % regime to the insertion of (some) cholesterol groups into the phospholipid monolayer. At higher surface concentrations the efficiency of insertion is reduced due to steric effects. We discuss the experimental results in the light of the energetics of the insertion of a cholesterol molecule into a lipid bilayer.