Rapid colorimetric detection of antibody-epitope recognition at a biomimetic membrane interface.

Biomolecular recognition of antigens and epitopes by antibodies is a fundamental event in the initiation of immune response and plays a central role in a variety of biochemical processes. Peptide binding requires, in many cases, presentation of the peptides at interfaces, such as protein surfaces, cellular membranes, and synthetic polymer surfaces. We describe a novel molecular system in which interactions between antibodies and peptide epitopes displayed at a biomimetic membrane interface can be detected through induction of visible, rapid color transitions. The colorimetric assembly consists of a phospholipid/polydiacetylene matrix anchoring a hydrophobic peptide displaying the epitope at its N-terminus. The colorimetric transitions observed in the assembly, corresponding to perturbation of the polydiacetylene framework, are induced only upon recognition of the displayed epitope by its specific antibody present in the aqueous solution. Significantly, the color changes occur after a single mixing step, without further chemical reactions or enzymatic processing. The new molecular system could be utilized for studying antigen-antibody interactions and peptide-protein recognition, epitope mapping, and rapid screening of biological and chemical libraries.     

Click Chemistry Mediated Functionalization of Vertical Nanowires for Biological Applications

Semiconductor nanowires (NWs) are gaining significant importance in various biological applications, such as biosensing and drug delivery. Efficient and controlled immobilization of biomolecules on the NW surface is crucial for many of these applications. Here, we present for the first time the use of the Cu^I‐catalyzed alkyne–azide cycloaddition and its strain‐promoted variant for the covalent functionalization of vertical NWs with peptides and proteins. The potential of the approach was demonstrated in two complementary applications of measuring enzyme activity and protein binding, which is of general interest for biological studies. The attachment of a peptide substrate provided NW arrays for the detection of protease activity. In addition, green fluorescent protein was immobilized in a site‐specific manner and recognized by antibody binding to demonstrate the proof‐of‐concept for the use of covalently modified NWs for diagnostic purposes using minute amounts of material.     

Peptide conformations for a microarray surface-tethered epitope of the tumor suppressor p53

Peptides or proteins near surfaces exhibit different structural properties from those present in a homogeneous solution, and these differences give rise to varied biological activity. Therefore, understanding the detailed molecular structure of these molecules tethered to a surface is important for interpreting the performance of the various microarrays based on the activities of the immobilized peptides or proteins. We performed molecular dynamics simulations of a pentapeptide, RHSVV, an epitope of the tumor suppressor protein p53, tethered via a spacer on a functionalized silica surface and free in solution, to study their structural and conformational differences. These calculations allowed analyses of the peptide-surface interactions, the sequence orientations, and the translational motions of the peptide on the surface to be performed. Conformational similarities are found among dominant structures of the tethered and free peptide. In the peptide microarray simulations, the peptide fluctuates between a parallel and tilted orientation driven in part by the hydrophobic interactions between the nonpolar peptide residues and the methyl-terminated silica surface. The perpendicular movement of the peptide relative to the surface is also restricted due to the hydrophobic nature of the microarray surface. With regard to structures available for recognition and binding, we find that similar conformations to those found in solution are available to the peptide tethered to the surface, but with a shifted equilibrium constant. Comparisons with experimental results show important implications of this for peptide microarray design and assays.     

Direct growth of shape-controlled nanocrystals on nanotubes via biological recognition

The new biological approach was examined to fabricate shape-controlled Ag nanocrystals grown directly on surfaces, inspired by nature that various shapes of nanocrystals are produced accurately and reproducibly in biological systems. Here we demonstrate the direct growth of hexagon-shaped Ag nanocrystals on sequenced peptide-coated nanotubes via biological recognition. When the peptide, Asn-Pro-Ser-Ser-Leu-Phe-Arg-Tyr-Leu-Pro-Ser-Asp, recognizing and effecting the Ag nanocrystal growth on the (111) face, was sequenced and incorporated onto template nanotube surfaces, the biomineralization of Ag ions on the nanotubes led the isotropic hexagon-shaped Ag nanocrystal coating under pH control of the growth solution. Multiple Ag nanocrystal shapes were observed when the peptide mineralized Ag ions without the template nanotubes, and therefore the template nanotube has a significant influence on regulating the majority of Ag nanocrystals into the hexagonal shape. This biological approach, using specific peptide sequences on surfaces to control nanocrystal shapes, may be developed as a simple and economical method to produce building blocks with desired physical properties for new generation of electronics, sensors, and optical devices.     

囊泡表面分子间通讯交流体系的构建: 利用受体的分子识别行为调控酶的活性

在人工双层膜囊泡表面, 构建了一个通过人工受体的分子识别行为控制酶反应活性的超分子体系. 体系以生物体细胞信号转导系统为模拟原型, 由作为受体的烷基胺、被受体识别的信号分子吡哆醛衍生物、乳酸脱氢酶、受体和酶之间的媒介物Cu^2＋以及作为体系载体的合成肽脂囊泡五个成分构成．通过UV-vis光谱法及动态光散射测定对体系进行了评价, 结果表明: 随着受体疏水参数增大, 其对信号分子的识别能力增强, 二者呈良好的线性关系; 通过信号分子与囊泡表面静电相互作用的研究表明信号分子具有选择性; 媒介物与信号分子–受体可形成化学计量比为1∶2的配合物, 其形成能力比媒介物与酶的结合能力更强．作为结论, 体系中烷基胺受体对磷酸吡哆醛信号分子的识别有效控制了处于囊泡表面的乳酸脱氢酶的活性．     

Artificial receptors that provides a preorganized hydrophobic environment: a biomimetic approach to dopamine recognition in water

[structure: see text] The recognition of dopamine in water has been achieved with tripodal oxazoline-based artificial receptors, capable of providing a preorganized hydrophobic environment by rational design, which mimics a hydrophobic pocket predicted for a human D2 receptor. The receptors show an amphiphilic nature owing to the presence of hydrophilic sulfonate groups at the periphery of the tripodal oxazoline ligands, which seems to contribute in forming the preorganized hydrophobic environment. The artificial receptors recognized dopamine hydrochloride in water with reasonable selectivity among various organoammonium guests examined. The observed binding behavior of the receptors was explained by evoking guest inclusion in the preorganized hydrophobic pocket-like environment and not by simple ion-pairing interactions. The rationally predicted 1:1 inclusion binding mode was supported by binding studies such as with a reference receptor that cannot provide a similar binding pocket, Job and VT-NMR experiments, electrospray ionization mass analysis, and guest selectivity data. This study implies that an effective hydrophobic environment can be generated even from an acyclic, small molecular artificial receptor. Such a preorganized hydrophobic environment, as being utilized in biological systems, can be effectively used as a complementary binding force for the recognition of organoammonium guests such as dopamine hydrochloride in water.     

Au nanowire fabrication from sequenced histidine-rich peptide

A new biological approach to fabricate Au nanowires was examined by using sequenced histidine-rich peptide nanowires as templates. The sequenced histidine-rich peptide molecules were assembled as nanowires, and the biological recognition of the sequenced peptide toward Au lead to efficient Au coating on the nanowires. Monodisperse Au nanocrystals were uniformly coated on the histidine peptide nanowires with the high-density coverage, and the crystalline phases of the Au nanocrystals were observed as (111) and (220). The uniformity of the Au coating on the nanowires without contamination of precipitated Au aggregates is advantageous for the fabrication of electronics and sensor devices when the nanowires are used as the building blocks. We believe this simple metal nanowire fabrication method can be applied to various metals and semiconductors with peptides whose sequences are known to mineralize specific ions.     

Isolation of ZnO-binding 12-mer peptides and determination of their binding epitopes by NMR spectroscopy

Inorganic-binding peptides are in the focus of research fields such as materials science, nanotechnology, and biotechnology. Applications concern surface functionalization by the specific coupling to inorganic target substrates, the binding of soluble molecules for sensing applications, or biomineralization approaches for the controlled formation of inorganic materials. The specific molecular recognition of inorganic surfaces by peptides is of major importance for such applications. Zinc oxide (ZnO) is an important semiconductor material which is applied in various devices. In this study the molecular fundamentals for a ZnO-binding epitope was determined. 12-mer peptides, which specifically bind to the zinc- or/and the oxygen-terminated sides of single-crystalline ZnO (0001) and (000-1) substrates, were selected from a random peptide library using the phage display technique. For two ZnO-binding peptides the mandatory amino acid residues, which are of crucial importance for the specific binding were determined with a label-free nuclear magnetic resonance (NMR) approach. NMR spectroscopy allows the identification of pH dependent interaction sites on the atomic level of 12-mer peptides and ZnO nanoparticles. Here, ionic and polar interaction forces were determined. For the oxygen-terminated side the consensus peptide-binding sequence (HSXXH) was predicted in silico and confirmed by the NMR approach.     

Preparing molecularly imprinted nanoparticles of saponins via cooperative imprinting strategy

Saponin is an important class of natural products with various pharmacological activities. The selective separation of saponins is an essential step before further analysis. Molecular imprinting has been an effective strategy for preparing antibody mimics. However, a facile and efficient imprinting strategy for saponins is still lacking owing to their amphiphilic nature. Herein, we have prepared the saponins imprinted nanoparticles via cooperative imprinting strategy. This new strategy relies on the combination of various non‐covalent interactions (hydrophobic and hydrogen bonding) and covalent boronate affinity interactions. The obtained imprinted nanoparticles could rebind specific saponins from complex matrices with good selectivity, superb tolerance to interference, and fast binding equilibrium. This method was verified to be versatile and facile. Thus, this strategy could greatly facilitate the preparation of imprinted nanoparticles for the specific recognition of saponins.     

Aryl Crown Ethers Based on d-Glucose Scaffold – Highly Selective Receptors of Amino Acid Ester Hydrochlorides

Amino acids are important biomolecules with a broad scope of applications in chemical and biological sciences. Their functions and properties depend on their absolute configuration. Therefore, methods for chiral recognition and separation of amino acids are highly sought after. For the purposes of diagnostic and medicinal applications chiral recognition of amino acids in water is particularly relevant. However synthetic receptors for enantioselective binding of amino acids in aqueous media are rare. Recently we reported a d -glucose-based crown ether for chiral recognition of amino acid esters in water. We achieved enantioselectivities towards amino acids with hydrophobic sidechains which were among the highest ever reported for a small molecule receptor. The binding affinities were however moderate. Herein we disclose analogs of that receptor, containing aryl functionalities in the crown ether fragment. The new receptors show considerably improved binding affinities for amino acid ester hydrochlorides in water, while retaining high enantio- or chemoselectivities.     

Advances in chiral separations of small peptides by capillary electrophoresis and chromatography

Many chemical and biological processes are controlled by the stereochemistry of small polypeptides (di‐, tri‐, tetra‐, penta‐, hexapeptides, etc). The biological importance of peptide stereoisomers is of great value. Therefore, the chiral resolution of peptides is an important issue in biological and medicinal sciences and drug industries. The chiral resolutions of peptide racemates have been discussed with the use of capillary electrophoresis and chromatographic techniques. The various chiral selectors used were polysaccharides, cyclodextrins, Pirkle types, macrocyclic antibiotics, crown ethers, imprinted polymers, etc. The stereochemistry of dipeptides is also discussed. Besides, efforts are made to explain the chiral recognition mechanisms, which will be helpful in understanding existing and developing new stereoselective analyses. Future perspectives of enantiomeric resolution are also predicted. Finally, the review concludes with the demand of enantiomeric resolution of all naturally occurring and synthetic peptides.     

Design criteria for engineering inorganic material-specific peptides

Development of a fundamental understanding of how peptides specifically interact with inorganic material surfaces is crucial to furthering many applications in the field of nanobiotechnology. Herein, we report systematic study of peptide sequence-activity relationships for binding to II-VI semiconductors (CdS, CdSe, ZnS, ZnSe) and Au using a yeast surface display system, and we define criteria for tuning peptide affinity and specificity for these material surfaces. First, homohexapeptides of the 20 naturally occurring amino acids were engineered, expressed on yeast surface, and assayed for the ability to bind each material surface in order to define functional groups sufficient for binding. Histidine (H6) was able to mediate binding of yeast to the five materials studied, while tryptophan (W6), cysteine (C6), and methionine (M6) exhibited different levels of binding to single-crystalline ZnS and ZnSe and polycrystalline Au surfaces. The ability of neighboring amino acids to up- and down-modulate histidine binding was then evaluated by use of interdigitated peptides (XHXHXHX). While the 20 amino acids exhibited a unique fingerprint of modulation for each material, some general trends emerged. With neutral defined by alanine, up-modulation occurred with glycine, basic amino acids, and the previously defined binding amino acids histidine, tryptophan, cysteine, and methionine, and down-modulation generally occurred with acidic, polar, and hydrophobic residues. We conclude that certain amino acids directly bind the material surface while neighboring amino acids locally modulate the binding environment for the materials we studied. Therefore, by the specific placement of up- and down-modulating amino acids, material specificity can be controlled. Finally, by employing the compositional and spatial criteria developed herein, it was possible to predictively design peptide sequences with material specificity, including a multimaterial binder, a Au-specific binder, and a ZnS-specific binder, that were verified as such in the context of yeast display.     

Photomodulated Azoaldolase: A Model for Light Intervention in Biological Systems?

Azoaldolase is obtained from rabbit muscle aldolase by adding an azo chromophore to a cysteine side chain in each of the four enzyme subunits. The enzyme becomes photosensitive whereas both its catalytic activity and the michaelian kinetics are retained. Chromophore excitation causes E to Z isomerization of the azo bond, and mutually influences the protein-substrate equilibria. The various isomerization and substrate binding equilibria have been investigated under the hypothesis of a cyclic process described by four linked equilibrium constants. The mechanism of the light effect is a continuous adaptation of the specific parameters of the active protein, that is substrate recognition and rate of the catalyzed process. Absorbed light allows the rapid modification of the concentrations of various related molecules, depending on the used frequencies. At present such a mechanism has not been described in photobiology; so azoaldolase can be taken as a model for a possible new mechanism of light regulation of a biological system, based on changes in the molecular recognition by an active protein against its substrate.     

Self-assembly of model DNA-binding peptide amphiphiles

Peptide amphiphiles combine the specific functionality of proteins with the engineering convenience of synthetic amphiphiles. These molecules covalently link a peptide headgroup, typically from an active fragment of a larger protein, to a hydrophobic alkyl tail. Our research is aimed at forming and characterizing covalently stabilized, self-assembled, peptide-amphiphile aggregates that can be used as a platform for the examination and modular design and construction of systems with engineering biological activity. We have studied the self-assembly properties of a model DNA-binding amphiphile, having a GCN4 peptide as the headgroup and containing a polymerizable methacrylic group in the tail region, using a combination of small-angle X-ray scattering, small-angle neutron scattering, and cryo- transmission electron microscopy. Our results reveal a variety of morphologies in this system. The peptide amphiphiles assembled in aqueous solution to helical ribbons and tubules. These structures transformed into lamella upon DNA binding. In contrast with common surfactants, the specific interaction between the headgroups seems to play an important role in determining the microstructure. The geometry of the self-assembled aggregate can be controlled by means of adding a cosurfactant. For example, the addition of SDS induced the formation of spherical micelles.     

Investigations of metal-coordinated peptides as supramolecular synthons

This article describes the synthesis and controlled assembly of four model biological-hybrid scaffolds via coordination of a metal complex to four new tripeptides. Each model tripeptide investigated has either a central pyridyl glycyl or a pyridyl alanyl residue between two terminally protected glycines. All tripeptides were coordinated to their complementary recognition unit, a p-methoxy SCS-Pd pincer complex. The assembly events were fully characterized and investigated by 1H NMR, ES-MS, and isothermal titration calorimetry (ITC) to elucidate how the substitution and spatial distance of the pyridyl moiety to the peptide backbone affects the metal coordination. Using these characterization techniques, we have shown that the metal-coordination events in all cases are fast and quantitative and that the peptide backbones do not interfere with the self-assembly. The ITC analyses showed that the 4-pyridyl tripeptides are the tightest binding ligands toward the palladated pincer complexes with the alanyl derivative being the strongest overall, demonstrating the superiority of the 4-pyridyl peptides over their 3-pyridyl analogues. The measured association constants are comparable to other pincer-pyridine systems in DMSO suggesting that the controlled coordination of the metalated pincer/pyridine interaction is an interesting biological synthon and will allow for the future development of important noncovalent peptide-based hybrid materials.     

Macrocyclic Receptors for Identification and Selective Binding of Substrates of Different Nature

Molecular recognition of host/guest molecules represents the basis of many biological processes and phenomena. Enzymatic catalysis and inhibition, immunological response, reproduction of genetic information, biological regulatory functions, the effects of drugs, and ion transfer—all these processes include the stage of structure recognition during complexation. The goal of this review is to solicit and publish the latest advances in the design and sensing and binding abilities of porphyrin-based heterotopic receptors with well-defined geometries, the recognition ability of which is realized due to ionic, H-bridge, charge transfer, hydrophobic, and hydrophilic interactions. The dissection of the considered low-energy processes at the molecular scale expands our capabilities in the development of effective systems for controlled recognition, selective delivery, and prolonged release of substrates of different natures (including drugs) to their sites of functioning.     

Context-dependent fluorescence detection of a phosphorylated tyrosine residue by a ribonucleopeptide

Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.     

Using combinatorial methods to arrive at a quantitative structure-stability relationship for a new class of one-armed cationic peptide receptors targeting the C-terminus of the amyloid beta-peptide

A new class of one-armed tripeptide based cationic guanidiniocarbonyl pyrrole receptors is shown to strongly bind the tetrapeptide L-Val-L-Val-L-Ile-L-Ala, representing the C-terminus of the amyloid beta-peptide even under polar conditions. A medium sized combinatorial library of 125 receptors was synthesized on a solid support and their binding properties determined on bead using a quantitative fluorescence assay. The binding constants are in the order of 10(3)-10(4) M-1 (in the presence of a formate counter ion in methanol) for the most efficient ones but differ by more than a factor of 100 among the 125 library members. Based on the binding data of 12 receptors a structure-stability relationship was established for peptide binding by this new receptor class. Complex formation is controlled by a fine balanced interplay of hydrophobic and electrostatic interactions with none of these two interactions alone being strong enough to ensure complexation under these polar conditions.     

DNA Framework-Based Topological Cell Sorters

Molecular recognition in cell biological process is characterized with specific locks-and-keys interactions between ligands and receptors, which are ubiquitously distributed on cell membrane with topological clustering. Few topologically-engineered ligand systems enable the exploration of the binding strength between ligand-receptor topological organization. Herein, we generate topologically controlled ligands by developing a family of tetrahedral DNA frameworks (TDFs), so the multiple ligands are stoichiometrically and topologically arranged. This topological control of multiple ligands changes the nature of the molecular recognition by inducing the receptor clustering, so the binding strength is significantly improved (ca. 10-fold). The precise engineering of topological complexes formed by the TDFs are readily translated into effective binding control for cell patterning and binding strength control of cells for cell sorting. This work paves the way for the development of versatile design of topological ligands.