High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.     

Rational disruption of the oligomerization of the mini-ferritin E. coli DPS through protein-protein interface mutation

DNA-binding protein from starved cells (DPS), a mini-ferritin capable of self-assembling into a 12-meric nano-cage, was chosen as the basis for an alanine-shaving mutagenesis study to investigate the importance of key amino acid residues, located at symmetry-related protein-protein interfaces, in controlling protein stability and self-assembly. Nine mutants were designed through simple inspection, synthesized, and subjected to transmission electron microscopy, circular dichroism, size exclusion chromatography, and "virtual alanine scanning" computational analysis. The data indicate that many of these residues may be hot spot residues. Most remarkably, two residues, R83 and R133, were observed to shift the oligomerization state to ~50% dimer. Based on the hypothesis that these two residues constitute a "hot strip," located at the ferritin-like threefold axis, the double mutant was generated which completely shuts down detectable formation of 12-mer in solution, favoring a cooperatively folded dimer. The fact that this effect logically builds upon the single mutants emphasizes that complex self-assembly has the potential to be manipulated rationally. This study should have an impact on the fundamental understanding of the assembly of DPS protein cages specifically and protein quaternary structure in general. In addition, as there is much interest in applying these and similar systems to the templation of nano-materials and drug delivery, the ability to control this ferritin's oligomerization state and stability could prove especially valuable.     

Zero and second‐derivative synchronous fluorescence spectroscopy for the quantification of two non‐classical β‐lactams in pharmaceutical vials: Application to stability studies

The formation of metal chelates with various ligands may lead to the production of fluorescent chelates or enhance the fluorescence of the chelating agent. This paper describes two sensitive, selective and computer‐solved methods, namely, zero order (SF) and second‐derivative synchronous spectrofluorimetry (SDSFS) for nano‐quantitation of two carbapenems; meropenem (MP) and ertapenem (EP). The methods are based on the chelation of MP with Tb³⁺ and EP with Zr⁴⁺ in buffered organic medium at pH 4.0 to produce fluorescent chelates. In the zero order method, the relative synchronous fluorescence intensity is measured at 327.0 nm at Δλ = 70.0 and 100.0 nm for MP and EP, respectively. The second method utilizes a second‐derivative technique to enhance the method selectivity and emphasize a stability‐indicating approach. The peak amplitudes (²D) of the second‐derivative synchronous spectra were estimated to be 333.06 and 330.06 nm for MP and EP, respectively. The proposed synchronous spectrofluorimetric methods were validated according to the International Conference on Harmonization (ICH) guidelines and applied successfully for the analysis of MP and EP in pure forms, pharmaceutical vials and in synthetic mixtures with different degradants of both drugs. Under optimum conditions, the mole‐ratio method was applied and the co‐ordination ratios of MP–Tb³⁺ and EP–Zr⁴⁺chelates were found to be 1:1 and 1:3. The formation constants for the chelation complexes were evaluated using the Benesi–Hildebrand's equation; the free energy change (ΔG) was also calculated. The results indicated that EP–Zr⁴⁺ was more stable than the MP–Tb³⁺ chelate. Moreover, the developed methods were found to be selective and inexpensive for quantitative determination of both drugs in quality control laboratories at nano‐levels.     

Graphene Wrapped FeSe2 Nano‐Microspheres with High Pseudocapacitive Contribution for Enhanced Na‐Ion Storage

Pseudocapacitance is a Faradaic process that involves surface or near surface redox reactions. Increasing the pseudocapacitive contribution is one of the most effective means to improve the rate performance of electrode materials. In this study, graphene oxide is used as a template to in situ synthesize burr globule‐like FeSe₂/graphene hybrid (B‐FeSe₂/G) using a facile one‐step hydrothermal method. Structural characterization demonstrates that graphene layers not only wrap the surfaces of FeSe₂ particles, but also stretch into the interior of these particles, as a result of which the unique nano‐microsphere structure is successfully established. When serving as anode material for Na‐ion batteries, B‐FeSe₂/G hybrid displays high electrochemical performance in the voltage range of 0.5–2.9 V. The B‐FeSe₂/G hybrid has high reversible capacity of 521.6 mAh·g^?1 at 1.0 A g^?1. Meanwhile, after 400 cycles, high discharge capacity of 496.3 mAh g^?1 is obtained at a rate of 2.5 A g^?1, with a high columbic efficiency of 96.6% and less than 1.0% loss of discharge capacity. Even at the ultrahigh rate of 10 A g^?1, a specific capacity of 316.8 mAh g^?1 can be achieved. Kinetic analyses reveal that the excellent performance of the B‐FeSe₂/G hybrid is largely attributed to the high pseudocapacitive contribution induced by the special nano‐micro structure.     

The neuropeptidome of Rhodnius prolixus brain

We show a sensitive and straightforward off‐line nano‐LC‐MALDI‐MS/MS workflow that allowed the first comprehensive neuropeptidomic analysis of an insect disease vector. This approach was applied to identify neuropeptides in the brain of Rhodnius prolixus, a vector of Chagas disease. This work will contribute to the annotation of genes in the ongoing R. prolixus genome sequence project. Peptides were identified by de novo sequencing and comparisons to known neuropeptides from different organisms by database search. By these means, we were able to identify 42 novel neuropeptides from R. prolixus. The peptides were classified as extended FMRF‐amide‐related peptides, sulfakinins, myosuppressins, short neuropeptide F, long neuropeptide F, SIF‐amide‐related peptides, tachykinins, orcokinins, allatostatins, allatotropins, calcitonin‐like diuretic hormones, corazonin, and pyrokinin. Some of them were detected in multiple isoforms and/or truncated fragments. Interestingly, some of the R. prolixus peptides, as myosuppressin and sulfakinins, are unique in their characteristic C‐terminal domain among insect neuropeptides identified so far.     

Exploring cellular adhesion and differentiation in a micro‐/nano‐hybrid polymer scaffold

Polymer scaffolds play an important role in three dimensional (3‐D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano‐fibrous and micro‐porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro‐scale pores and nano‐scale fibers. To evaluate the advantages of micro/nano combination, control scaffolds with only micro‐pores or nano‐fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro‐ and nano‐scale structures. The NFMP scaffold nano‐fibers promoted neural differentiation and induced “3‐D matrix adhesion”, while the NFMP scaffold micro‐pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro‐only, nano‐only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Prospects for using a new sequential chemiluminescence strategy for monitoring the caffeine content in soft and energy drinks via the catalytic activities of different nano‐metal oxides

This article suggests a new sequential injection analysis chemiluminescence (SIA‐CL) strategy for monitoring the caffeine (CAF) content in soft and energy drinks using the catalytic activities of different nano‐metal oxides. The present study describes three different SIA‐CL systems (luminol–ferricyanide (III) coupled with Fe₂O₃ or ZnO nanoparticles (NPs), and luminol–H₂O₂ coupled with CuONPs. All experimental conditions were optimized and the linear concentration ranges of pure CAF were evaluated using the calibration graphs. The selectivity of the developed SIA‐CL systems was studied under the influence of various interfering species that may be present in soft or energy drinks such as sodium ions, sucrose, glucose, sodium benzoate, sodium citrate, riboflavin, niacin, citric, phosphoric and ascorbic acids. International Council for Harmonization (ICH) guidelines were obeyed for the validation of the suggested CL methods. The developed SIA‐CL systems displayed linear relationships over the concentration ranges 1.0–350, 5.0–400 and 10.0–400 μg ml^?1 with Fe₂O₃ NPs, ZnO NPs and CuO NPs, respectively. The recorded lower limits of detection and quantification were 0.7, 2.7 and 7.8 μg ml^?1, and 1.0, 5.0 and 10.0 μg ml^?1 for the previously mentioned SIA‐CL systems. The results revealed high selectivity for CAF determination and were in good agreement with those obtained by other reported methods.     

Study on syntheses and anticoagulant action of heparin/rare earth nano‐oxides hybrid material

Four hybrid materials of RE₂O₃‐TDI‐Heparin (TDI = Toluene 2,4–diisocyanate, RE = La, Eu, Nd, Sc) were prepared by the method of graft. The materials were characterized by IR, TG, and SEM, which confirmed that the heparin was grafted on the surface of TDI modified rare earth nano‐oxides. The cell adhesion experiment and the anticoagulant experiment demonstrated that the materials have lower cell toxicity, better cell adhesion as well as better anticoagulant action. In addition, the clotting time of hybrid materials were shortened compared with the heparin. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 887–892, 2010.     

Preparation,characterization and luminescent properties of dense nano‐silica hybrids loaded with 1,8‐naphthalic anhydride

Novel luminescent dense nano‐silica hybrid materials (DNSS) modified with different amounts of (3‐aminopropyl)triethoxysilane (APTES) and 1,8‐naphthalic anhydride (NA) were successfully synthesized via two steps combined with post‐grafting methods. Powder X‐ray diffraction (XRD), N₂‐sorption analysis, Fourier transform infrared (FT‐IR) spectroscopy, transmission electron microscopy (TEM), thermogravimetric analysis (TGA), photoluminescence (PL) spectroscopy and elemental analysis, as well as time‐resolved decays were employed to characterize the resultant hybrid materials. The results revealed that luminescent organic molecules had been successfully loaded onto the amine‐modified surface of nano‐silica spheres. In addition, their fluorescence intensity and characteristic peak of emission spectra changed with increasing amount of APTES and NA additive. In particular, the characteristic peak showed a red shift from 390 to 450 nm, however, this was inconsistent with results calculated on the basis of the elemental analysis data, most probably because of the dispersion behaviors of NA molecules from the aggregating to the monolayer state. These observations demonstrated the existence of a quantum confinement effectiveness of NA–DNSS samples, and therefore a possible mechanism was put forward. Copyright © 2013 John Wiley & Sons, Ltd.     

Cathodic electrochemiluminescent behavior of luminol at nafion-nano-TiO2 modified glassy carbon electrode

The electrochemiluminescence (ECL) behavior of luminol on a nafion-nano-TiO(2) modified glassy carbon electrode (nafion-nano-TiO(2)--GCE) was studied. Two ECL peaks (ECL-1 and ECL-2) were found during cathodic potential scanning. ECL-1 at ca -0.4 V (vs Ag--AgCl reference electrode) came from the reaction between luminol and active oxygen anion produced at the GCE surface directly, while ECL-2 at ca -0.9 V (vs Ag--AgCl reference electrode) came from the reaction between luminol and the active oxygen anion catalyzed by TiO(2.) The possible mechanism for the generation of both ECL peaks has been proposed. The reproducibility of the ECL intensities on nafion-nano-TiO(2)--GCE at ECL-1 and ECL-2 was good, with relative standard deviations (n = 10) of 4.3 and 1.3%, respectively. The ECL-2 generated at the nafion-nano-TiO(2)--GCE surface was further developed to detect the dissolved oxygen, and a detection limit of 0.02 mg/L was achieved. The proposed method was applied to detect dissolved oxygen in water with satisfactory result.     

Metal‐dependent assembly of a protein nano‐cage

Short, alpha‐helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo–designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion‐dependent assembly of a tetrahedral protein cage. Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺ ions were evaluated, with Ni²⁺ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion–protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.     

Effects of vibration on differentiation of cultured PC12 cells

Different types of physiological‐mechanical stress, such as shear stress in vascular endothelial cells or hydrostatic pressure in chondrocytes are well known as regulators of cell function. In this study, the effects of vibration, a type of non‐physiological mechanical stimulation, on differentiation of rat pheochromocytoma (PC12) cells are reported. A nano‐vibration system was designed to produce nanometer‐scale vibration. The frequency and amplitude of the nano‐vibrations were monitored by a capacitance displacement sensor connected to an oscilloscope. When PC12 cells exposed to nerve growth factor were subjected to vibration at 10 kHz, differentiation and elongation of their neurites were promoted earlier in the culture. Vibration promoted differentiation of PC12 cells. This approach could therefore also be promising for determining of the effects of the physical environment on cell differentiation. Biotechnol. Bioeng. 2011; 108:592–599. © 2010 Wiley Periodicals, Inc.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Synthesis of nano‐sized hydrogen phosphate‐imprinted polymer in acetonitrile/water mixture and its use as a recognition element of hydrogen phosphate selective all‐solid state potentiometric electrode

Herein, a new recipe is introduced for the preparation of hydrogen phosphate ion‐imprinted polymer nanoparticles (nano‐IIP) in acetonitrile/water (63.5:36.5) using phosphoric acid as the template. The nano‐IIP obtained was used as the recognition element of a carbon paste potentiometric sensor. The IIP electrode showed a Nernstian response to hydrogen phosphate anion; whereas, the non‐imprinted polymer (NIP)‐based electrode had no considerable sensitivity to the anion. The presence of both methacrylic acid and vinyl pyridine in the IIP structure, as well as optimization of the functional monomers‐template proportion, was found to be important to observe the sensing capability of the IIP electrode. The nano‐IIP electrode showed a dynamic linear range of 1 × 10^?5‐1 × 10^?1 mol L‐1, Nernstian slope of 30.6 ± (0.5) mV decade ^?1, response time of 25 seconds, and detection limit of 4.0 × 10^?6 mol L^?1. The utility of the electrodes was checked by potentiometric titration of hydrogen phosphate with La³⁺ solution.