Selective Protein Recognition Research Articles

Selective protein recognition in supported lipid bilayer arrays by tailored, dual-mode deep cavitand hosts.

Self-folding deep cavitands with variably functionalized upper rims are able to selectively immobilize proteins at a biomimetic supported lipid bilayer surface. The immobilization process takes advantage of the dual-mode binding capabilities of the hosts, combining a defined binding pocket with upper rim charged/H-bonding groups. A variety of proteins can be selectively immobilized at the bilayer interface, either via complementary charge/H-bonding interactions, cavity-based molecular recognition, or a combination of both. The immobilization process can be used to bind unmodified native proteins, epitopes for bioadhesion, or proteins covalently modified with suitable RNMe3+ binding "handles" and charged groups that can either match or mismatch with the cavitand rim. The immobilization process can be monitored in real time using surface plasmon resonance (SPR) spectroscopy, and applied to the construction of cavitand:lipid arrays using the hosts and trehalose vitrified phospholipid vesicles. The selective, dual-mode protein recognition is maintained in the arrays, and can be visualized using SPR imaging.

Fabrication of imidazolium-functionalized magnetic composite microspheres for selective recognition and separation of heme proteins

Imidazolium-functionalized magnetic composite microspheres were successfully synthesized, which exhibited high hemoglobin binding capacity (6321 mg g−1) and excellent selectivity.

Detection of Intracellular Selenol-Containing Molecules Using a Fluorescent Probe with Near-Zero Background Signal

Selenocysteine (Sec), encoded as the 21st amino acid, is the predominant chemical form of selenium that is closely related to various human diseases. Thus, it is of high importance to identify novel probes for sensitive and selective recognition of Sec and Sec-containing proteins. Although a few probes have been reported to detect artificially introduced selenols in cells or tissues, none of them has been shown to be sensitive enough to detect endogenous selenols. We report the characterization and application of a new fluorogenic molecular probe for the detection of intracellular selenols. This probe exhibits near-zero background fluorescence but produces remarkable fluorescence enhancement upon reacting with selenols in a fast chemical reaction. It is highly specific and sensitive for intracellular selenium-containing molecules such as Sec and selenoproteins. When combined with flow cytometry, this probe is able to detect endogenous selenols in various human cancer cells. It is also able to image endogenous selenol-containing molecules in zebrafish under a fluorescence microscope. These results demonstrate that this molecular probe can function as a useful molecular tool for intracellular selenol sensing, which is valuable in the clinical diagnosis for human diseases associated with Sec-deficiency or overdose.

RNF4-mediated SUMOylation is essential for NDRG2 suppression of lung adenocarcinoma.

N-Myc downstream-regulated gene 2 (NDRG2) protein is a tumor suppressor that inhibits cancer growth, metastasis and invasion. The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin through its selective recognition and ubiquitination of SUMO-modified proteins. We evaluated NDRG2 SUMOylation in lung adenocarcinoma cells and its underlying molecular mechanism. The results showed that NDRG2 is covalently modified by SUMO1 at K333, which suppressed anchorage independent adenocarcinoma cell proliferation and tumor growth. In human lung adenocarcinomas cells, RNF4 targeted NDRG2 to proteasomal degradation by stimulating its SUMOylation. Endogenous RNF4 expression was increased in human lung adenocarcinomas cells, and there was a concomitant upregulation of SUMO. These findings indicate that SUMOylation of NDRG2 is necessary for its tumor suppressor function in lung adenocarcinoma and that RNF4 increases the efficiency of this process.

Bio-inspired magnetic molecularly imprinted polymers based on Pickering emulsions for selective protein recognition

Hydrophobic halloysite nanotube stabilized Pickering emulsions and their application in protein separation.

Cell and Protein Recognition at a Supported Bilayer Interface via In Situ Cavitand-Mediated Functional Polymer Growth.

Water-soluble deep cavitands embedded in a supported lipid bilayer are capable of anchoring ATRP initiator molecules for the in situ synthesis of primary amine-containing polymethacrylate patches at the water:membrane interface. These polymers can be derivatized in situ to incorporate fluorescent reporters, allow selective protein recognition, and can be applied to the immobilization of nonadherent cells at the bilayer interface.

Super-Paramagnetic Nanoparticles by Surface Imprinting on Graphene Oxide Modified Iron (II, III) with Application for the Determination of Ovalbumin by Absorption Spectroscopy

Protein surface imprinting produces materials capable of selective recognition and capture of proteins. Herein, a protein surface imprinted polymer on graphene oxide modified super-paramagnetic Fe3O4 nanoparticles is reported. The molecularly imprinted polymer was synthesized by ultrasound-assisted suspension polymerization, using ovalbumin as the template molecule, 3-aminophenylboronie acid as the functional monomer, and methylene-bis-acrylamide as the cross-linking agent. The nanoparticles were approximately 40 nanometers in size and super-paramagnetic. Moreover, these particles demonstrated considerably high adsorption capacity, fast adsorption kinetics, and selective binding affinities toward the template protein ovalbumin. The calibration curve of ovalbumin was linear from 5.0 × 10−11 to 1.0 × 10−10 molar. The limit of detection of ovalbumin was 2.0 × 10−11 M. These results show that this super-paramagnetic material has potential for biological macromolecule separation and determination.

Aptamer-based organic-silica hybrid affinity monolith prepared via “thiol-ene” click reaction for extraction of thrombin

A novel strategy for preparing aptamer-based organic-silica hybrid monolithic column was developed via “thiol-ene” click chemistry. Due to the large specific surface area of the hybrid matrix and the simplicity, rapidness and high efficiency of “thiol-ene” click reaction, the average coverage density of aptamer on the organic-silica hybrid monolith reached 420pmolμL−1. Human α-thrombin can be captured on the prepared affinity monolithic column with high specificity and eluted by NaClO4 solution. N-p-tosyl-Gly-Pro-Arg p-nitroanilide acetate was used as the sensitive chromogenic substrate of thrombin. The thrombin enriched by this affinity column was detected with a detection of limit of 0.01μM by spectrophotometry. Furthermore, the extraction recovery of thrombin at 0.15μM in human serum was 91.8% with a relative standard deviation of 4.0%. These results indicated that “thiol-ene” click chemistry provided a promising technique to immobilize aptamer on organic–inorganic hybrid monolith and the easily-assembled affinity monolithic material could be used to realize highly selective recognition of trace proteins.

Protein imprinting over magnetic nanospheres via a surface grafted polymer for specific capture of hemoglobin

Combining surface molecular imprinting with a nano-sized physical form is an effective technology to achieve a large binding capacity and overcome template removal within molecularly imprinted polymers (MIPs), and is in particular advantageous to the template of bulky structures like proteins. In addition, the surface protein-imprinted polymers immobilized on nanospheres with magnetic function are more attractive in the research of selective recognition and enrichment of proteins. In this work, we have demonstrated a facile method, enhancing specific recognition of multifunctional molecularly imprinted magnetic nanospheres with core–shell structure. It was achieved simply via radical induced graft polymerization of low concentration monomers on the surface of vinyl groups modified magnetic nanospheres dispersed in aqueous media using hemoglobin (Hb) as a template. The possible gelation of the dispersion after polymerization was avoided and hence the imprinted magnetic particles without an obvious agglomerated phenomenon could be collected readily and expediently. Finally, a highly monodisperse magnetic nanospheres were synthesized successfully. The morphological studies of the resultant MIPs-coated magnetic particles were characterized using various methods. In batch rebinding tests, the imprinted magnetic particles reached saturated adsorption within 30 min and exhibited significant specific recognition towards the target protein molecules with a binding capacity as high as 281.94 mg g−1 and an imprinting factor of 4.29. The resultant Hb-MIPs magnetic particles not only extracted target protein from mixed proteins, but also enriched the low abundance of hemoglobin. Moreover, the stability and regeneration were also investigated, which indicated the excellent property of reutilization.

Membrane traffic research: challenges for the next decade.

The study of membrane traffic is now a well-established area of research, and one that has resulted in several Nobel prizes including ones awarded to Albert Claude, George Palade, and Christian DeDuve in 1974, Michael Brown and Joseph Goldstein in 1985, Gunter Blobel in 1999, and most recently James Rothman, Randy Schekman, and Thomas Sudhof in 2013. As a result of their studies and other research, we now have fundamental insights into the organization and routes of transport between the cells' membranous organelles. Moreover, we have defined the basic “cellular machinery” that governs protein and lipid synthesis, that ensures selective recognition of proteins and lipids, and that promotes vesicle fission, transport, and fusion. In addition, we have a large number of insights into the regulatory molecules that control these processes including the Rab GTPases and their effectors. While the challenges for the future are many, this essay is focused on areas of investigation that we see as moving forward at a rapid pace, which speak to how membrane traffic contributes to overall cell and tissue function, and which are likely to provide important avenues of funding for both established and new investigators. These challenges include how membrane traffic is regulated in response to metabolic needs, how molecules are transferred between organelles, how membrane traffic is regulated and functions during processes such as development, and how membrane traffic is used by highly differentiated cells to perform specialized cell functions.

RNF 4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

Lithography: Nanosphere Lithography as a Versatile Method to Generate Surface‐Imprinted Polymer Films for Selective Protein Recognition (Adv. Funct. Mater. 37/2013)

Lithography: Nanosphere Lithography as a Versatile Method to Generate Surface‐Imprinted Polymer Films for Selective Protein Recognition (Adv. Funct. Mater. 37/2013)

Polyanion cluster patterning on polymer surface through microemulsion approach for selective adsorption of proteins

A facile method to fabricate honeycomb-patterned polymer films bearing cavities that are locally decorated with inorganic component is developed in this study. By mixing poly(methyl methacrylate) dichloromethane solution containing P123 with polyoxometalate (POM) aqueous solution through shaking, a reversed hybrid microemulsion is obtained. The evaporation of solvent in the microemulsion on solid surface yields an ordered porous film accompanied by the accumulation of P123 and POMs on the inner surface of the cavities. The formation of patterned structure is proved to be independent from the type of POMs, but the size of the cavities can be adjusted to some extent by changing the concentration of surfactant and polymer, and the volume ratio of water and dichloromethane in the solution used for casting. The locally anchored POMs can be readily applied for the selective recognition of proteins. BSA and hemoglobin patterns are then fabricated through their electrostatic interactions with POMs. At lower pH, POM pattern could prior recognize hemoglobin from its mixed solution of BSA, generating a characteristic pattern. The reported work creates an efficient way of patterning organically incompatible components, such as water-soluble molecules and nanoparticles, on porous polymer films for the fabrication of multi-functional hybrid surface structures.

Low-Fouling, Biospecific Films Prepared by the Continuous Assembly of Polymers

We report that the continuous assembly of polymers (CAP) approach, mediated by ring-opening metathesis polymerization (ROMP), is a facile and versatile technology to prepare engineered nanocoatings for various biomedical applications. Low-fouling coatings on particles were obtained by the formation of multicompositional, layered films via simple and efficient tandem CAP(ROMP) processes that are analogous to chain extension reactions. In addition, the CAP(ROMP) approach allows for the efficient postfunctionalization of the CAP films with bioactive moieties via cross-metathesis reactions between the surface-immobilized catalysts and symmetrical alkene derivatives. The combined features of the CAP(ROMP) approach (i.e., versatile polymer selection and facile functionalization) allow for the fabrication and surface modification of various types of polymer films, including those with intrinsic protein-repellent properties and selective protein recognition capabilities. This study highlights the various types of advanced coatings and materials that the CAP approach can be used to generate, which may be useful for biomedical applications.

Nanosphere Lithography as a Versatile Method to Generate Surface‐Imprinted Polymer Films for Selective Protein Recognition

AbstractA versatile approach based on nanosphere lithography is proposed to generate surface‐imprinted polymers for selective protein recognition. A layer of 750 nm diameter latex bead‐protein conjugate is deposited onto the surface of gold‐coated quartz crystals followed by the electrosynthesis of a poly(3,4‐ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) film with thicknesses on the order of the bead radius. The removal of the polymer bead‐protein conjugates, facilitated by using a cleavable protein‐nanosphere linkage is shown to result in 2D arrays of periodic complementary size cavities. Here it is demonstrated by nanogravimetric measurements that the imprinting proceeds further at molecular level and the protein (avidin) coating of the beads generates selective recognition sites for avidin on the surface of the PEDOT/PSS film. The binding capacity of such surface‐imprinted polymer films is ca. 6.5 times higher than that of films imprinted with unmodified beads. They also exhibit excellent selectivity against analogues of avidin, i.e., extravidin, streptavidin, and neutravidin, the latter being in fact undetectable. This methodology, if coupled with properly oriented conjugation of the macromolecular template to the nanoparticles, offers the possibility of site‐directed imprinting.

Protein‐Directed Synthesis of Mn‐Doped ZnS Quantum Dots: A Dual‐Channel Biosensor for Two Proteins

Proteins typically have nanoscale dimensions and multiple binding sites with inorganic ions, which facilitates the templated synthesis of nanoparticles to yield nanoparticle-protein hybrids with tailored functionality, water solubility, and tunable frameworks with well-defined structure. In this work, we report a protein-templated synthesis of Mn-doped ZnS quantum dots (QDs) by exploring bovine serum albumin (BSA) as the template. The obtained Mn-doped ZnS QDs give phosphorescence emission centered at 590 nm, with a decay time of about 1.9 ms. A dual-channel sensing system for two different proteins was developed through integration of the optical responses (phosphorescence emission and resonant light scattering (RLS)) of Mn-doped ZnS QDs and recognition of them by surface BSA phosphorescent sensing of trypsin and RLS sensing of lysozyme. Trypsin can digest BSA and remove BSA from the surface of Mn-doped ZnS QDs, thus quenching the phosphorescence of QDs, whereas lysozyme can assemble with BSA to lead to aggregation of QDs and enhanced RLS intensity. The detection limits for trypsin and lysozyme were 40 and 3 nM, respectively. The selectivity of the respective channel for trypsin and lysozyme was evaluated with a series of other proteins. Unlike other protein sensors based on nanobioconjugates, the proposed dual-channel sensor employs only one type of QDs but can detect two different proteins. Further, we found the RLS of QDs can also be useful for studying the BSA-lysozyme binding stoichiometry, which has not been reported in the literature. These successful biosensor applications clearly demonstrate that BSA not only serves as a template for growth of Mn-doped ZnS QDs, but also impacts the QDs for selective recognition of analyte proteins.

ChemInform Abstract: Small Targeted Cytotoxics: Current State and Promises from DNA‐Encoded Chemical Libraries

AbstractReview: 234 refs.

Small Targeted Cytotoxics: Current State and Promises from DNA‐Encoded Chemical Libraries

The targeted delivery of potent cytotoxic agents has emerged as a promising strategy for the treatment of cancer and other serious conditions. Traditionally, antibodies against markers of disease have been used as drug-delivery vehicles. More recently, lower molecular weight ligands have been proposed for the generation of a novel class of targeted cytotoxics with improved properties. Advances in this field crucially rely on efficient methods for the identification and optimization of organic molecules capable of high-affinity binding and selective recognition of target proteins. The advent of DNA-encoded chemical libraries allows the construction and screening of compound collections of unprecedented size. In this Review, we survey developments in the field of small ligand-based targeted cytotoxics and show how innovative library technologies will help develop the drugs of the future.

Protein recognition on a single graphene oxide surface fixed on a solid support

We study protein recognition on a graphene oxide (GO) surface using a single GO piece fixed on a solid support. The GO surface is modified by newly designed processes using pyrene as a linker to an sp2 domain in the GO, an aptamer for thrombin recognition, and a probe dye for fluorescence detection. In this system, the dye probe fluorescence, which was initially quenched by GO, is recovered when the aptamer recognizes the corresponding protein. We demonstrate the label-free and selective protein recognition for thrombin. The elementary processes of protein recognition are observed directly with a confocal laser scanning microscope and an atomic force microscope using an identical piece of GO. They indicate that proteins are recognized homogeneously on the modified GO surface. We also show that the recognition system can be installed and operated in microchannel devices.

Direct observation of selective protein capturing on molecular imprinting substrates

A sensing interface for specific protein capture was fabricated using a novel molecular imprinting (MIP) process. Bovine serum albumin (BSA) and ovalbumin (OVA) were imprinted on a quartz substrate with modified alkyl groups, and target protein capture was detected using a deep-UV fluorescence image microscope (UVFLIM). The imprinted protein was immobilized to silica beads (diameter: 15μm) using a phospholipid polymer containing both active ester groups and silane coupling groups, which were used as protein stamps to prepare the imprinting surface. Protein recognition sites were constructed by integrating sodium dodecyl sulfate (SDS) as the ligand, which was immobilized with a biocompatible photoreactive phospholipid polymer. When BSA solution was added to the BSA-based MIP substrate, strong fluorescence was observed from the tryptophan residue of BSA. In contrast, for the OVA-based MIP substrate and non-MIP substrate, no fluorescence was observed. The surface showed good selectivity of BSA against OVA. The phospholipid polymer layer prevented non-specific protein adsorption, resulting in highly selective protein recognition. Further, when the protein-imprinted substrate was constructed without ligands, neither protein was captured on the substrate. We demonstrated the importance of ligand integration for capturing target proteins at specific positions. UVFLIM can be used to detect biomolecules at the single-molecule level by using intrinsic fluorescence without molecular labeling. Our new protein-imprinted surface used with UVFLIM is a versatile tool for capturing biomolecules.