Nanoscale patterning of solid-supported membranes by integrated diffusion barriers

Ultraflat nanostructured substrates have been used as a template to create patterned solid-supported bilayer membranes with polymerizable tethered lipids acting as diffusion barriers. Patterns in the size range of 100 nm were successfully produced and characterized. The diffusion barriers were embedded directly into the phospholipid bilayer and could be used to control the fluidity of the membrane as well as to construct isolated membrane corrals. By using nanosphere lithography to structure the templates it was possible to systematically adjust the lipid diffusion coefficients in a range comparable to those observed in cellular membranes. Single colloids applied as mask in the patterning process yielded substrates for creation of isolated fluid membrane patches corralled by diffusion barriers. Numerous potential applications for this new model system can be envisioned, ranging from the study of cellular interactions or of molecular diffusion in confined geometries to biosensor arrays.     

Electrochemical growth of highly oriented organic-inorganic superlattices using solid-supported multilamellar membranes as templates

Controllable depositing of relatively thick inorganic sublayers into organic templates to fabricate organic-inorganic superlattices is of great importance. We report a novel approach to fabricating phospholipid/Ni(OH)(2) superlattices by electrochemical deposition of the inorganic component into solid-supported multilamellar templates. The well-ordered and highly oriented multilamellar templates are produced by spreading small drops of lipid solution on silicon surfaces and letting the solvent evaporate slowly. The templates which are used as working electrodes preserve the lamellar structure in the electrolyte solution. The resulting superlattices are highly oriented. The thickness of the nickel hydroxide is controlled by the concentration of nickel ions in the electrolyte bath. The electron density profiles derived from the X-ray diffraction data reveal that the thickness of the nickel hydroxide sublayers increases from 15 to 27 A as the concentration of nickel nitrate increases from 0.005 mol/L to 0.08 mol/L. We expect that the new method can be extended to depositing a variety of inorganic components including metals, oxides, and semiconductors.     

Self-assembly of solid-supported membranes using a triggered fusion of phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane

A general procedure for the formation ofsolid-supported artificial membranes containing transmembrane proteins is reported. The main objective was to directly use the pool of proteins of the native biomembrane (here the inner membrane from mitochondria of human carcinogenic hepatic cells) and to avoid purification steps with detergent. Proteoliposomes of phospholipid-enriched inner membranes from mitochondria were tethered and fused onto a tailored surface via a streptavidin link. The failure of some preliminary experiments on membrane formation was attributed to strong nonspecific interactions between the solid surface and the protuberant hydrophilic parts of the transmembrane complexes. The correct loading of uniform membranes was performed after optimization of a tailored surface, covered with a grafted short-chain poly(ethylene glycol), so that nonspecific interactions are reduced. Step-by-step assembly of the structure and triggered fusion of the immobilized proteoliposomes were monitored by surface plasmon resonance and fluorescence photobleaching recovery, respectively. The long-range lateral diffusion coefficient (at 22 degrees C) for a fluorescent lipid varies from 2.5 x 10(-8) cm2 s(-1) for a tethered lipid bilayer without protein to 10(-9) cm2 s(-1) for a tethered membrane containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The decrease in the diffusion coefficient in the tethered membrane with increase in protein area fraction was too pronounced to be fully explained by the theoretical models of obstructed lateral diffusion. Covalent tethering links with the solid are certainly involved in the decrease of the overall lateral mobility of the components in the supported membrane at the highest protein-to-lipid ratios.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Molecular self-assembly of solid-supported protein crystals

Highly ordered protein arrays have been proposed as a means for templating the organization of nanomaterials. Toward this end, we investigate the ability of the protein streptavidin to self-assemble into various configurations on solid-supported phospholipids. We identify two genetic variants of streptavidin (comprising amino acids 14-136 and 13-139) and examine their molecular organization at the liquid-solid interface. Our results demonstrate that the structural differences between these two protein variants affect both crystalline lattice and domain morphology. In general, these results for the liquid-solid interface are similar and consistent with those at the air-water interface with a few notable differences. Analogous to crystallization at the air-water interface, both forms of streptavidin yield H-like domains with lattice parameters that have C222 symmetry at pH 7. At pH 4, the native, truncated form of streptavidin yields needle-like domains consisting of molecules arranged in P1 symmetry. Unlike crystalline domains grown at the air-water interface, however, the lattice parameters of this P1 crystal are unique and have not yet been reported. The presence of a solid substrate does not appear to dramatically alter streptavidin's two-dimensional crystallization behavior, suggesting that local intermolecular interactions between proteins are more significant than interactions between the interface and protein. Our results also demonstrate that screening the electrostatic repulsion between protein molecules by modulating ionic strength will increase growth rate while decreasing crystalline domain size and macroscopic defects. Finally, we show that these domains are indeed functional by attaching biotinylated gold nanoparticles to the crystals. The ability to modulate molecular configuration, crystalline defects, and domain size on a functional array supports the potential application of this system toward materials assembly.     

Controlled delivery of proteins into bilayer lipid membranes on chip

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.     

Electrophoretic transfer of proteins across polyacrylamide membranes

The electrophoretic transfer of purified proteins has been examined in a Gradiflow "Babyflow BF100" unit. A number of factors affect protein separation within this preparative electrophoresis system. We established that the rate of protein transfer was proportional to the applied voltage. The transfer is slowest at the isoelectric point (pI) and increased the further away the pH was from the pI of the protein. Protein transfer was found to be independent of the ionic strength of the buffer, for buffers that excluded the addition of strong acids or strong bases or sodium chloride. Transfer decreased as the pore size of the membrane decreased. Finally, transfer was inhibited at high salt concentrations in the protein solution, but remained unaffected when urea and non-ionic detergents were added to the solution. To increase the speed of protein separations, buffers with low conductivity should be used. A pH for the optimal separation should be selected on the basis of the relative pI and size of the target proteins and that of the major contaminants.     

Charge-coupled device camera-based detection of fluorescence-labeled proteins immobilized on nitrocellulose membranes

Proteins dotted on nitrocellulose membranes are biotinylated by reaction with a biotinyl-succinimide ester. The resulting biotinyl residues serve as specific binding sites for a subsequent streptavidin-based detection system. Using streptavidin-peroxidase, the proteins are visualized either by deposition of a colored formazan dye or by enhanced chemiluminescence the latter being twofold more sensitive. Alternatively, streptavidin-fluorescein isothiocyanate (FITC) is substituted for the peroxidase conjugate as tool for protein staining. The sensitivity of both staining variants is dramatically improved by the inclusion of the reporter deposition technique. The fluorescence-labeled proteins are visualized on a visible blue light emitting illuminator preventing the bothering effect of photobleaching. In combination with a charge-coupled device (CCD) camera-based image analyzing system the established stain with streptavidin-FITC detects about 10 pg of protein dot blotted on nitrocellulose membranes.     

Electric field effects in proteins in membranes

Both the organization and function of protein nanostructures in membranes are related to the substructural properties of the lipid portion of the membrane. Potential differences that are established across the membrane and generate electric fields in these very thin portions are shown to modulate the organizational and functional properties of the protein modules. Many protein modules also have nonisotropic distributions of charged sites, including configurations in which there are regions containing predominantly positive fixed charges, juxtaposed with adjacent regions containing predominantly negative fixed charges. In these double fixed charge regions, very large electric fields can manifest in the ionic depletion layer at the junction of the two fixed charge regions.Consideration is also given to the manner in which the intense electric fields that are established in protein modules, such as proton ATPases, can modulate the chemical reactions that are associated with proton transport and dehydration reactions.     

Suzuki cross-coupling of solid-supported chloropyrimidines with arylboronic acids

The utility of the Suzuki cross-coupling to synthesize biaryl compounds is expanded herein to include reactions of resin-supported chloropyrimidines with boronic acids. In particular, an efficient method is described for the synthesis of a library of biaryl compounds from solid-supported chloropyrimidines. The Suzuki reaction was performed in an inert atmosphere using Pd(2)(dba)(3)/P(t-Bu)(3) as catalyst, spray-dried KF as base, and THF as solvent. The reaction was allowed to proceed overnight at 50 degrees C. Upon cleavage with acid, a library of 4-(substituted amino)-6-arylpyrimidines was obtained in moderate yield and high purity.     

Calculations of pH-dependent binding of proteins to biological membranes

Binding of proteins to membranes is often accompanied by titration of ionizable residues and is, therefore, dependent on pH. We present a theoretical treatment and computational approach for predicting absolute, pH-dependent membrane binding free energies. The standard free energy of binding, DeltaG, is defined as -RTln(P(b)/P(f)), where P(b) and P(f) are the amounts of bound and free protein. The apparent pK(a) of binding is the pH value at which P(b) and P(f) are equal. Proteins bind to the membrane in the pH range where DeltaG is negative. The components of the binding free energy are (a) the free energy cost of ionization state changes (DeltaG(ion)), (b) the effective energy of transfer from solvent to the membrane surface, (c) the translational/rotational entropy cost of binding, and (d) an ideal entropy term that depends on the relative volume of the bound and free state and therefore depends on lipid concentration. Calculation of the first term requires determination of pK(a) values in solvent and on the membrane surface. All energies required by the method are obtained from molecular dynamics trajectories on an implicit membrane (IMM1-GC). The method is tested on pentalysine and the helical peptide VEEKS, derived from the membrane-binding domain of phosphocholine cytidylyltransferase. The agreement between the measured and the calculated free energies of binding of pentalysine is good. The extent of membrane binding of VEEKS is, however, underestimated compared to experiment. Calculations of the interaction energy between two VEEKS helices on the membrane suggest that the discrepancy is mainly due to the neglect of protein-protein interactions on the membrane surface.     

Formation of solid-supported lipid bilayers: an integrated view

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential bio-technological applications. A qualitative understanding of the process of SLB formation after exposure of small lipid vesicles to a hydrophilic support is now emerging. Recent studies have revealed a stunning variety of effects that can take place during this self-organization process. The ensemble of results in our group has revealed unprecedented insight into intermediates of the SLB-formation process and has helped to identify a number of parameters that are determinant for the lipid deposition on solid supports. The pathway of lipid deposition can be tuned by electrostatic interactions and by the presence of calcium. We emphasize the importance of the solid support in the SLB-formation process. Our results suggest that the molecular-level interaction between lipids and the solid support needs to be considered explicitly, to understand the rupture of vesicles and the formation of SLBs as well as to predict the properties of the resulting SLB. The impact of the SLB-formation process on the quality and the physical properties of the resulting SLB as well as implications for other types of surface-confined lipid bilayers are discussed.     

Improved method for the diimide reduction of multiple bonds on solid-supported substrates

A mild and improved method for reducing multiple bonds on various resins with diimide is described. The simple procedure readily generates diimide from 2-nitrobenzenesulfonohydrazide and triethylamine at room temperature. A number of representative multiple bonds in various steric and electronic environments were examined, including polar double bonds such as carbonyl and azo, for ease and selectivity of reduction. A general trend of reactivity was identified which revealed, inter alia, that terminal olefins, 1,2-disubstituted olefins, electron-poor olefins, and terminal alkynes were the most easily reduced.     

A traceless solid-supported synthesis of novel pyrazinediazepinedione derivatives

A simple, convenient, six-step synthesis of novel, tricyclic pyrazinebenzodiazepinedione derivatives has been described. The strategy is based on the use of the orthogonally-protected, optically pure, (S)-piperazine-2-carboxylic acid, in a Petasis reaction, followed by coupling with anthranilic acid and finally cyclizing cleavage. The investigated method was applied for the synthesis of novel bicyclic pyrazinediazepinedione derivatives. This traceless, solid-supported approach allows the preparation of a wide variety of compounds in moderate yields from commercially available or easily obtainable reagents.     

Targeting proteins to liquid-ordered domains in lipid membranes

We demonstrate the construction of novel protein-lipid assemblies through the design of a lipid-like molecule, DPIDA, endowed with tail-driven affinity for specific lipid membrane phases and head-driven affinity for specific proteins. In studies performed on giant unilamellar vesicles (GUVs) with varying mole fractions of dipalymitoylphosphatidylcholine (DPPC), cholesterol, and diphytanoylphosphatidyl choline (DPhPC), DPIDA selectively partitioned into the more ordered phases, either solid or liquid-ordered (L(o)) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the L(o) phase containing DPIDA. In the presence of CuCl(2), the iminodiacetic acid (IDA) headgroup of DPIDA forms the Cu(II)-IDA complex that exhibits a high affinity for histidine residues. His-tagged proteins were bound specifically to domains enriched in DPIDA, demonstrating the capacity to target protein binding selectively to both solid and L(o) phases. Steric pressure from the crowding of surface-bound proteins transformed the domains into tubules with persistence lengths that depended on the phase state of the lipid domains.