Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation.

Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

Design of RGDS Peptide-Immobilized Self-Assembling ß-Strand Peptide from Barnacle Protein.

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Strategy to Immobilize Peptide Probe Selected through In Vitro Ribosome Display for Electrochemical Aptasensor Application.

In this study, we demonstrated an electrochemical aptasensor for calmodulin (CaM) detection and the peptide sequence (YWDKIKDFIGG) is obtained from in vitro ribosome display selection. To immobilize this peptide probe on the electrode surface, cystine was incorporated at the end of this peptide sequence. After a maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EODT-MI), film was electropolymerized on the electrode, the peptide probe was immobilized through thiol-ene conjugation with the cystine end. Four peptides with different linkers were used for the binding test of bovine serum albumin and CaM using a quartz crystal microbalance. The zwitterionic linker EKEKEKEKEKEK provided good antifouling properties and the highest CaM binding. Furthermore, the immobilization of the peptide with this zwitterionic linker resulted in a minimal increase in the electrochemical impedance. By immobilizing the peptide with the selected zwitterionic linker, we successfully demonstrated an electrochemical aptasensor with a linear detection range for CaM from 0.01 to 10 mg/L and a detection limit of 0.001 mg/L.

Selective Immobilization of Fluorescent Proteins for the Fabrication of Photoactive Materials.

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

Purification-independent immunoreagents obtained by displaying nanobodies on bacteria surface.

The availability of preimmune libraries of antibody fragments allows for the fast generation of binders which can be expressed in both eukaryotic and prokaryotic systems. We exploited the recombinant nature of antibody fragments to demonstrate the possibility of expressing them as functional proteins displayed on the surface of Escherichia coli and by such a way to generate living reagents ready-to-use for diagnostics. Such immunoreagents were effectively exploited without the necessity of any purification step to prepare immunocapture surfaces suitable for the diagnostic of both cancer cells and toxic microalgae. The same nanobody-displaying bacteria were also engineered to coexpress GFP in their cytoplasm. Suspensions of such living fluorescent immunoreagents effectively bound to eukaryotic cells making them visible and quantifiable by flow cytometry analysis and using 96-well plate readers. The collected data showed the suitability of such living immunoreagents for reproducible and inexpensive diagnostic applications.

In situ removal of consensus dengue virus envelope protein domain III fused to hydrophobin in Pichia pastoris cultures.

This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed. The protein was produced and purified directly from the yeast culture supernatant both efficiently and with no loss. The purified protein was properly immobilized by adsorption in solid phase and recognized by anti-dengue antibodies, showing its potential for the development of an indirect immunoassay for dengue virus.

Antibody-Binding, Antifouling Surface Coatings Based on Recombinant Expression of Zwitterionic EK Peptides.

Development of antifouling films which selectively capture or target proteins of interest is essential for controlling interactions at the "bio/nano" interface. However, in order to synthesize biofunctional films from synthetic polymers that incorporate chemical "motifs" for surface immobilization, antifouling, and oriented biomolecule attachment, multiple reaction steps need to be carried out at the solid/liquid interface. EKx is a zwitterionic peptide that has previously been shown to have excellent antifouling properties. In this study, we recombinantly expressed EKx peptides and genetically encoded both surface attachment and antibody-binding motifs, before characterizing the resultant biopolymers by traditional methods. These peptides were then immobilized to organosilica nanoparticles for binding IgG, and subsequently capturing dengue NS1 as a model antigen from serum-containing solution. We found that a mixed layer of a short peptide (4.9 kDa) "backfilled" with a longer peptide terminated with an IgG-binding Z-domain (18 kDa) demonstrated selective capture of dengue NS1 protein down to ∼10 ng mL-1 in either PBS or 20% serum.

Biosynthesis of Thermoresponsive Magnetic Nanoparticles by Magnetosome Display System.

Thermoresponsive magnetic nanoparticles (MNPs) were synthesized using a magnetosome display system. An elastin-like polypeptide decamer of VPGVG (ELP10), which is hydrophobic above the transition temperature ( Tt) and can form an insoluble aggregation, was immobilized on biogenic MNPs in the magnetotactic bacterium, Magnetospirillum magneticum AMB-1. It was suggested that hydrophobicity of the MNP surface increased at 60 °C compared with 20 °C by the immobilization of ELP10. Size distribution analysis indicated that the immobilization of ELP10 onto MNPs induced the increased hydrophobicity with increasing temperatures up to 60 °C, promoting aggregation of the particles by hydrophobic and magnetic interactions. These results suggest that the acceleration of magnetic collection at 60 °C was caused by particle aggregation promoted by hydrophobic interaction between ELP-MNPs. Furthermore, the immobilization of ELP on MNPs gave a quick magnetic collection at 60 °C by external magnetic field. The thermoresponsive properties will further expand the utility of biotechnological applications of biogenic MNPs.

ATP binding and hydrolysis disrupt the high-affinity interaction between the heme ABC transporter HmuUV and its cognate substrate-binding protein.

Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, e.g. vitamin B12, heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the Yersinia pestis ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism.

Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor-α.

To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts. Graphical Abstract ᅟ.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) immobilized on laser-fabricated 3D scaffolds enhance osteogenesis.

The regeneration of bone via a tissue engineering approach involves components from the macroscopic to the nanoscopic level, including appropriate 3D scaffolds, cells and growth factors. In this study, hexagonal scaffolds of different diagonals were fabricated by Direct Laser Writing using a photopolymerizable hybrid material. The proliferation of bone marrow (BM) mesenchymal stem cells (MSCs) cultured on structures with various diagonals, 50, 100, 150 and 200µm increased significantly after 10days in culture, however without significant differences among them. Next, recombinant human bone morphogenetic protein 2 (rhBMP-2) was immobilized onto the hybrid material both via covalent binding and physical adsorption. Both immobilization types exhibited similar high releaseate bioactivity profiles and a sustained delivery of rhBMP-2. The collagen and calcium levels produced in the extracellular matrix (ECM) were significantly elevated for the samples functionalized with BMP-2 compared to those in the osteogenic medium. Furthermore, significant upregulation of gene expression in both types of BMP-2 immobilized scaffolds was observed for alkaline phosphatase (ALPL) and osteocalcin (BGLAP) at days 7, 14, and 21, for RUNX2 at day 21, and for osteonectin (SPARC) at days 7 and 14. The results suggest that the release of bioactive rhBMP-2 from the hybrid scaffolds enhance the control over the osteogenic differentiation during cell culture.

Simple surface functionalization of polymersomes using non-antibacterial peptide anchors.

BACKGROUND: Hollow vesicles formed from block copolymers, so-called polymersomes, have been extensively studied in the last decade for their various applications in drug delivery, in diagnostics and as nanoreactors. The immobilization of proteins on the polymersomes' surface can aid in cell targeting, lead to functional biosensors or add an additional reaction space for multistep syntheses. In almost all surface functionalization strategies to date, a chemical pre-conjugation of the polymer with a reactive group or ligand and the functionalization of the protein are required. To avoid chemical pre-conjugation, we investigated the simple and quick functionalization of preformed poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) polymersomes through the spontaneous insertion of four hydrophobic, non-antibacterial peptide anchors into the membrane to display enhanced green fluorescent protein (eGFP) on the polymersomes' surface. RESULTS: Three of the four hydrophobic peptides, the transmembrane domains of a eukaryotic cytochrome b 5 , of the viral lysis protein L and of the yeast syntaxin VAM3 could be recombinantly expressed as soluble eGFP-fusion proteins and spontaneously inserted into the polymeric membrane. Characterization of the surface functionalization revealed that peptide insertion was linearly dependent on the protein concentration and possible at a broad temperature range of 4-42 °C. Up to 2320 ± 280 eGFP molecules were immobilized on a single polymersome, which is in agreement with the calculated maximum loading capacity. The peptide insertion was stable without disrupting membrane integrity as shown in calcein leakage experiments and the functionalized polymersomes remained stable for at least 6 weeks. CONCLUSION: The surface functionalization of polymersomes with hydrophilic proteins can be mediated by several peptide anchors in a spontaneous process at extremely mild insertion conditions and without the need of pre-conjugating polymers.

Distal M domain of cobra ADAM-like metalloproteinase mediates the binding of positively charged cysteine-rich domain to αvß3 integrin in the suppression of cell migration.

We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture. Herein, we investigated their functional targets to elucidate the role of cobra SVMPs in perturbing wound healing in snakebite victims. We showed that the non-RGD (Arg-Gly-Asp) C-shaped SVMP atragin binds about ten-fold stronger than the RGD-containing I-shaped SVMP kaouthiagin-like to αvß3 integrin in the surface-immobilized form. Atragin binds to αvß3 integrin through a novel interaction mode involving distal M and C domains via the RRN sequence motif in the hyper variable loop. In a cell adhesion assay, the adhesion of fibroblasts to atragin was mediated by αvß3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in a αvß3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvß3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims.

Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin.

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.

A peptide derivative serves as a fibroblast growth factor 2 antagonist in human gastric cancer.

Fibroblast growth factor 2 (FGF2) plays a critical role in tumorigenesis and progression of solid tumor and is upregulated in gastric carcinoma serum. Therefore, it is regarded as a potential therapeutic target of human gastric cancer. Suppression of bioactivities of FGF2 may contribute to human gastric cancer therapy. Herein, we obtained a novel FGF2-binding peptide derivative (named P32), which originated from a previously isolated P7 peptide with poor stability. We proved that P32, which had a half-life in human plasma up to 12 h, enhanced stability and exerted strong inhibitory effect on FGF2-induced cell proliferation and invasion in human gastric cancer cell lines. Further investigations revealed that the underlying anti-proliferation mechanisms of P32 in vitro included arresting FGF2-stimulated cells at the G0/G1 phase and reducing the activation of AKT and Erk1/2 cascades. The FGF2-binding peptide derivative P32 has improved stability, is relatively safe, and may have therapeutic potential in FGF2-driven gastric cancer.

Microchip ELISA coupled with cell phone to detect ovarian cancer HE4 biomarker in urine.

Ovarian cancer is a leading cause of death from gynecologic cancers in the USA, and early diagnosis can potentially increase 5-year survival rate. Detection of biomarkers derived from hyperplasia of epithelial tissue by enzyme-linked immunosorbent assay (ELISA) proves to be a practical way of early diagnosis of ovarian cancer. However, ELISA is commonly performed in a laboratory setting, and it cannot be used in a clinical setting for on-site consultation. We have shown a microchip ELISA that detects HE4, an ovarian cancer biomarker, from urine using a cell phone integrated with a mobile application for imaging and data analysis. In microchip ELISA, HE4 from urine was first absorbed on the surface; the primary and secondary antibodies were subsequently anchored on the surface via immuno-reaction; and addition of substrate led to color development because of enzymatic labeling. The microchip after color development was imaged using a cell phone, and the color intensity was analyzed by an integrated mobile application. By comparing with an ELISA standard curve, the concentration of HE4 was reported on the cell phone screen. The presented microchip ELISA coupled with a cell phone is portable as opposed to traditional ELISA, and this method can facilitate the detection of ovarian cancer at the point-of-care (POC).

Controlling dissociation channels of gas-phase protein complexes using charge manipulation.

Coarse-grained simulations with charge hopping were performed for a positively charged tetrameric transthyretin (TTR) protein complex with a total charge of +20. Charges were allowed to move among basic amino acid sites as well as N-termini. Charge distributions and radii of gyration were calculated for complexes simulated at two temperatures, 300 and 600 K, under different scenarios. One scenario treated the complex in its normal state allowing charge to move to any basic site. Another scenario blocked protonation of all the N-termini except one. A final scenario used the complex in its normal state but added a basic-site containing tether (charge tag) near the N-terminus of one chain. The differences in monomer unfolding and charging were monitored in all three scenarios and compared. The simulation results show the importance of the N-terminus in leading the unfolding of the monomer units; a process that follows a zipper-like mechanism. Overall, experimentally modifying the complex by adding a tether or blocking the protonation of N-termini may give the potential for controlling the unraveling and subsequent dissociation of protein complexes.

Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-ß-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl ß-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Label-free fluorescent detection of thrombin activity based on a recombinant enhanced green fluorescence protein and nickel ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles.

Herein, a novel label-free fluorescent assay has been developed to detect the activity of thrombin and its inhibitor, based on a recombinant enhanced green fluorescence protein (EGFP) and Ni(2+) ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Ni(2+)-NTA MNPs). The EGFP, containing a thrombin cleavage site and a hexahistidine sequence (His-tag) at its N-terminal, was adsorbed onto Ni(2+)-NTA MNPs through Ni(2+)-hexahistidine interaction, and dragged out of the solution by magnetic separation. Thrombin can selectively digest EGFP accompanied by His-tag peptide sequence leaving, and the resulting EGFP cannot be captured by Ni(2+)-NTA MNPs and kept in supernatant. Hence the fluorescence change of supernatant can clearly represent the activity of thrombin. Under optimized conditions, such assay showed a relatively low detection limit (3.0×10(-4) U mL(-1)), and was also used to detect the thrombin inhibitor, Hirudin, and further applied to detect thrombin activity in serum. Combined with the satisfactory reusability of Ni(2+)-NTA MNPs, our method presents a promising candidate for simple, sensitive, and cost-saving protease activity detecting and inhibitor screening.

Conductometric monitoring of protein-protein interactions.

Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.