Directed supramolecular surface assembly of SNAP-tag fusion proteins

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized SNAP-fusion proteins on cyclodextrin-coated surfaces yielded stable monolayers. The binding of the fusion proteins is specific and occurs with an affinity in the order of 10(6) M(-1) as determined by surface plasmon resonance. Reversible micropatterns of the fusion proteins on micropatterned cyclodextrin surfaces were visualized by using fluorescence microscopy. Furthermore, the guest-functionalized proteins could be assembled out of solution specifically onto the surface of cyclodextrin vesicles. The SNAP-tag labeling of proteins thus allows for assembly of modified proteins through a host-guest interaction on different surfaces. This provides a new strategy in fabricating protein patterns on surfaces and takes advantage of the high labeling efficiency of the SNAP-tag with designed supramolecular elements.     

Protein-directed assembly of binary monolayers at the interface and surface patterns of protein on the monolayers

Ferritin-directed assembly of binary monolayers of zwitterionic dipalmitoylphosphatidylcholine and cationic dioctadecyldimethylammonium bromide (DOMA) at the interface and surface patterns of ferritin on the monolayers have been investigated using a combination of infrared reflection absorption spectroscopy, surface plasmon resonance, and atomic force microscopy. Ferritin binding to the binary monolayers at the air-water interface at the surface pressure 30 mN/m, primarily driven by the electrostatic interaction, gives rise to a change in tilt angle of hydrocarbon chains from 15 degrees +/- 1 degrees to 10 degrees +/- 1 degrees with respect to the normal of the monolayer at the mole fraction of DOMA (XDOMA) of 0.1. The chains at XDOMA = 0.3 are oriented vertical to the water surface before and after protein binding. A new mechanism for protein binding to the binary monolayers is proposed. The secondary structures of the adsorbed ferritin are prevented from changing to some extent due to the existence of the monolayers. The amounts of the bound protein on the monolayers at the air-water interface are increased in comparison with those on the pre-immobilized monolayers at low XDOMA. The increased amounts and different patterns of the adsorbed protein at the monolayers are mostly attributed to the formation of multiple binding sites available for ferritin, which is due to the lateral reorganization of the lipid components in the monolayers induced by the protein in the subphase. The created multiple binding sites on the monolayer surfaces through the protein-directed assembly can be preserved for subsequent protein binding.     

Controlled protein assembly on a switchable surface

The strategy presented in this work supplies a general method of controlling protein assembly on a switchable low-density SAM, which may open a new way to design functional biocomposite films for biosensors or protein chips.     

Submicron streptavidin patterns for protein assembly

Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure to 365 nm (i-line) light. Upon PAG activation and hydrolysis of acetals, aldehyde groups formed. After the films were incubated with a biotinylated aldehyde reactive probe, the X-ray photoelectron spectroscopy results were consistent with biotin being attached to the surface. The background was subsequently passivated by flood exposure and incubation with an aminooxy-terminated poly(ethylene glycol), resulting in a 98% reduction in nonspecific protein adsorption. Protein patterning and assembly was demonstrated using streptavidin, biotinylated anthrax toxin receptor-1, and the protective antigen moiety of anthrax toxin and confirmed by fluorescence microscopy and atomic force microscopy (AFM). AFM demonstrated that 500 nm protein features were achieved. Because of the abundance of biotinylated proteins, this methodology provides a platform for protein immobilization and assembly for various applications in biotechnology.     

Templated assembly of a pseudorotaxane of gold rectangles

A nanoscopic pseudorotaxane (1)2 subset2 composed of the gold rectangle [Au4(mu-PAnP)2(mu-bipy)2](OTf)4 and the linear template 4,4'-bis(9'-anthryl)biphenyl was assembled (PAnP = 9,10-bis(diphenylphosphino)anthracene); bipy = 4,4'-bipyridine).     

SNAP-tag蛋白标签技术与荧光分析法在蛋白原位分析中的联合应用

为将生物体内微观的蛋白行为可视化并以宏观信号呈现出来对蛋白进行实时、动态分析，借助SNAP-tag蛋白标签技术与有机小分子荧光染料，构建了一系列用于活细胞内实时监测目标蛋白的免洗荧光探针。标签蛋白SNAP-tag能够特异性识别探针中的苄基鸟嘌呤，从而使目标蛋白共价连接上荧光团（萘酰亚胺），携带上荧光信使。此外，由于萘酰亚胺从水环境中被牵引至SNAP-tag蛋白的疏水空腔，其荧光信号呈现出2~13倍的增强。通过SNAP-tag标签蛋白与目标蛋白的融合，该荧光探针实现了对活细胞内线粒体蛋白CoX8A及核内蛋白H2B特异性识别，在免洗条件下完成了对目标蛋白的实时追踪及原位分析。     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

Templated assembly of polymer particles into mesoscopic clusters with well-defined configurations

We report on the fabrication of colloidal clusters through the combination of spherical particles. Polystyrene latex particles bearing amino groups on their surface were used as building blocks of the clusters. Packing of these particles with diameters of 91 and 154 nm into assemblies with defined configurations was accomplished using narrow dispersed emulsion droplets as templates. The building blocks of the clusters adhered to the oil–water interphase due to the Pickering effect. Subsequent evaporation of the dispersed phase forced them to pack into small clusters. Addition of the particles via the dispersed phase led to higher yields of clusters than if the building blocks were added via the continuous phase. All clusters had well-defined configurations. Because the dimensions of these clusters were below 400 nm, the colloidal assemblies underlay Brownian motion, which resulted in stable suspensions. The number and yields of different species could be controlled via the concentration of the building blocks and surfactant within the emulsions. Moreover, the nature of the dispersed phase itself had a strong impact on the cluster formation. When cyclohexane was used as the dispersed phase, predominately, particle doublets and triplets were obtained. The use of toluene-in-water emulsions resulted into a broader spectrum of clusters of up to 12 constituents. Such clusters could satisfy the demand for particles with complex but defined shapes and special symmetries for the fabrication of novel hierarchically organized materials.     

Templated assembly of organic-inorganic materials using the core shell structure of the P22 bacteriophage

Biomimetic chemistry offers new approaches to supramolecular materials synthesis and assembly. We have demonstrated that an assembled viral protein cage, comprising an organic core-shell structure, can be used as a template for the size constrained synthesis of Fe(2)O(3). Particle nucleation is directed by the inner scaffold protein layer, while the size constraints are determined by the outer capsid layer.     

Templated assembly of sulfide nanoclusters into cubic-C3N4 type framework

Here we report a new type of nanocluster superlattice in which each four-connected cluster ([M4In16S31],6- M = Fe, Co, Zn, and Cd) alternates with a three-connected sulfur anion (S2-) to form a rare and yet theoretically important non-centrosymmetric and non-interpenetrating (3,4)-connected net topologically identical to that of the hypothetical cubic carbon nitride type net. These materials have a ring size consisting of 16 tetrahedral atoms. Because of the large cluster size and the elimination of structural intergrowth, the volume fraction of the inorganic framework is as low as 38%. A strong photoluminescent emission has also been observed.     

Templated construction of a Zn-selective protein dimerization motif

Here, we report that the approach of metal-templated ligand synthesis can be applied to construct a dimeric protein assembly ((BMOE)RIDC1(2)), which is stabilized by noncovalent interactions and flexible covalent cross-linkers around the Zn templates. Despite its flexibility, (BMOE)RIDC1(2) selectively binds Zn(II) over other divalent metals and undergoes dimerization upon metal binding. Such simultaneous fulfillment of plasticity and selectivity is a hallmark of cellular signaling events that involve ligand/metal-induced protein dimerization.     

Templated synthesis of silver nanowires based on the layer-by-layer assembly of silver with dithiodipropionic acid molecules as spacers

The layer-by-layer assembly of silver nanoclusters with 3,3'-dithiodipropionic acid (DTDPA) as spacers was prepared through self-assembly on a gold foil and has been characterized by cyclic voltammetric and AFM techniques. The DTDPA molecules acting as spacers between the layers of silver serve as molecular interconnects for the four layers prepared in this work. The organization of layers was found to decrease with an increase in the number of layers. The layer-by-layer assembly of silver clusters motivated us to prepare silver nanowires stabilized by the bifunctional molecules DTDPA through template synthesis using cellulose nitrate membranes. The nanostructures formed by this method were characterized by SEM, TEM, AFM, FTIR, CV, and photoluminescence studies. It is observed that the DTDPA molecules, instead of forming molecular interconnects, protect the structures by self-assembling themselves along the edges of the nanostructures. The concept of self-assembly protecting the nanostructures is demonstrated in this work.     

How to prevent the loss of surface functionality derived from aminosilanes

Aminosilanes are common coupling agents used to functionalize silica surfaces. A major problem in applications of 3-aminopropylsilane-functionalized silica surfaces in aqueous media was encountered: the loss of covalently attached silane layers upon exposure to water at 40 degrees C. This is attributed to siloxane bond hydrolysis catalyzed by the amine functionality. To address the issue of loss of surface functionality and to find conditions where hydrolytically stable amine-functionalized surfaces can be prepared, silanization with different types of aminosilanes was carried out. Hydrolytic stability of the resulting silane-derived layers was examined as a function of reaction conditions and the structural features of the aminosilanes. Silane layers prepared in anhydrous toluene at elevated temperature are denser and exhibit greater hydrolytic stability than those prepared in the vapor phase at elevated temperature or in toluene at room temperature. Extensive loss of surface functionality was observed in all 3-aminopropylalkoxysilane-derived layers, independent of the number and the nature of the alkoxy groups. The hydrolytic stability of aminosilane monolayers derived from N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES) indicates that the amine-catalyzed detachment can be minimized by controlling the length of the alkyl linker in aminosilanes.     

Hydrocarbon dispersions of acrylic microspheres with polar surface functionality

Submicron and micron sized particles containing a crosslinked core and a polar shell were prepared by 3-stage nonaqueous dispersion [NAD] polymerization in an aliphatic hydrocarbon medium. When a poly (12-hydroxystearic acid) [PHS] comb stabilizer was used in all three stages, the particles produced were spherical, submicron in size, and had a relatively broad size distribution. If the monomer mixture in the third stage contained relatively large amounts of butyl acrylate, stable dispersions of sponge-like aggregates, 3–5 μm in size, were obtained. When butyl rubber was employed as a stabilizer precursor in the seed formation step, the shape of the final particles depended upon whether a crosslinker was employed in the second stage polymerization. When a crosslinker was present, the second-stage particles and the subsequent third-stage particles had a shrunken, raisin-like appearance. When no crosslinker was present, spherical particles were obtained. In both cases, the resulting third-stage particles were easily dispersed in water containing a small amount of amine base, indicating that ? COOH and ? OH groups are located at the surface (or in a shell at the surface) in these NAD particles. © 1994 John Wiley & Sons, Inc.     

Bottom-up assembly of molecular wagons on a surface

The bottom-up assembly of molecular building blocks, carrying specific functions, is a promising strategy for the construction of nanomachines. In this study we show how molecules with a mechanical function, i.e., being equipped with wheels, can be connected in a controlled way directly on a surface. By choosing suitable building blocks, assembled dimers and wagon trains can be formed, whereas the length of the chains can be limited by using a heterogeneous mixture of molecules. By using low temperature scanning tunneling microscopy, the chemical nature of the intermolecular connection is determined as a metal-ligand bond, which is stable enough to maintain the wagon train structure at room temperature. The intermolecular bonds can be controllably changed from trans to cis configurations thereby achieving bond angles of almost 90°.     

Ligand functionality as a versatile tool to control the assembly behavior of preformed titania nanocrystals

Nanoparticle powders composed of surface-functionalized anatase crystals with diameters of about 3 nm self-organize into different structures upon redispersion in water. The assembly is directed by a small amount of a low-molecular-weight functional ligand (the "assembler") adsorbed on the surface of the nanoparticles. The ligand functionality determines the anisotropy of the resulting structures. Multidentate ligands, such as trizma ((HOCH(2))(3)CNH(2)) and serinol ((HOCH(2))(2)CNH(2)), with a chargeable terminal group preferentially induce the formation of anisotropic nanostructures several hundreds of nanometers in total length, whereas all the other investigated ligands (ethanolamine H(2)N(CH(2))(2)OH, glycine hydroxamate H(2)NCH(2)CONHOH, dopamine (OH)(2)C(6)H(3)(CH(2))(2)NH(3)Cl, tris (HOCH(2))(3)CCH(3)) mainly lead to uncontrolled agglomeration. Experimental data suggests that the anisotropic assembly is a consequence of the water-promoted desorption of the organic ligands from the {001} faces of the crystalline building blocks together with the dissociative adsorption of water on these crystal faces. Both processes induce the preferred attachment of the titania nanoparticles along the [001] direction. The use of polydentate and charged ligands to functionalize the surface of nanoparticles thus provides a versatile tool to control their arrangement on the nanoscale.     

Templated assembly of gold nanoparticles into microscale tubules and their application in surface-enhanced Raman scattering

We report a simple procedure to assemble gold nanoparticles into hollow tubular morphology with micrometer scale, wherein the citrate molecule is used not only as a reducing and capping agent, but also as an assembling template. The nanostructure and growth mechanism of microtubes are explored via SEM, TEM, FTIR spectra, and UV-vis spectra studies. The incorporation of larger gold nanoparticles by electroless plating results in an increase in the diameter of microtubes from 900 nm to about 1.2 microm. The application of the microtubes before and after electroless plating in surface-enhanced Raman scattering (SERS) is investigated by using 4-aminothiophenol (4-ATP) as probe molecules. The results indicate that the microtubes both before and after electroless plating can be used as SERS substrates. The microtubes after electroless plating exhibit excellent enhancement ability.     

Controlled assembly of bacteria on chemical patterns using soft lithography

Highly ordered arrays of single living bacteria were obtained by selective adsorption of bacteria onto chemical patterns with micrometric resolution. The chemically engineered template surfaces were prepared with the combination of microcontact printing process and a simple incubation technique. This methodology can be used for fundamental studies of bacterium's inner mechanisms and sub-cellular organization as well as for interfacing living bacteria with artificial microsystems.     

Fine-tuning the surface functionality of aqueous luminescent nanocrystals through surfactant bilayer modification

We report a two-step phase transfer approach to locate surfactant bilayers on water-soluble luminescent nanocrystals (NCs), through which the surface functionality of the NCs is tunable. Since the species of both inner and outer surfactants of the bilayer are alterable in wide range, the current effort provides a facile approach to anchor various functional groups on aqueous NCs. The primary results indicate that these bilayer-modified NCs are able to be incorporated with various organic and inorganic materials.     

A polymeric chiral surface functionality immobilized on uniformly sized macroporous polymer beads

Uniformly sized functionalized macroporous polymer beads were prepared by either a classical copolymerization method or recently reported in situ surface modification method utilizing chiral methacrylamide as a functional modifier. To evaluate conformational and/or specific differences in their surface chiral functionality, we applied chromatographic evaluation techniques. The prepared modified beads were utilized as chiral stationary phase in high-performance liquid chromatography (HPLC). Those prepared by the in situ surface modification method tended to show higher chiral recognition ability than those by the classical copolymerization method, even if the equivalent amount of the chiral functional group was involved within polymer beads. Detailed chromatographic studies exhibited the in situ surface modification method could lead to polymeric methacrylamide functionality on the surface within relatively large pore size regions of the macroporous polymer beads, while the classical copolymerization method tended to form less polymeric surface functionality. The difference in the chiral surface functionality on both of macroporous polymer beads afforded drastic change in chromatographic chiral recognition ability. Complete resolution of a drug, thalidomide could be achieved on the chiral stationary phase with the polymeric chiral surface functionality, while no resolution was found on that with the monomeric one even if the same chiral methacrylamide was used as a modifier to prepare the chiral stationary phases. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35 : 2747–2757, 1997