Green fluorescent protein for in situ synthesis of highly uniform Au nanoparticles and monitoring protein denaturation

Purified recombinant green fluorescent protein (GFP) expressed in E. coli was used for single-step synthesis of gold nanoparticles (Au NPs) with extraordinary size specificity in aqueous medium. The fluorescence of GFP offered a probe for concomitant changes in the protein during the course of synthesis, in addition to the monitoring of the time-dependent formation of Au NPs by the surface plasmon resonance. Reaction of AuCl⁻₄ with the protein produced spherical Au NPs having diameters ranging from 5–70 nm. Remarkably, addition of 1.0×10⁻⁵ M AgNO₃ in the medium produced uniform spherical Au NPs with particle diameter of 2.2±0.5 nm. Fluorescence spectroscopic measurements suggest that during synthesis of Au NPs in absence of AgNO₃, partial denaturation of the protein occurred resulting in the lowering of fluorescence intensity. On the other hand, when the NPs were synthesized in the presence of AgNO₃ complete denaturation of the protein with complete loss of fluorescence could be observed, which was further confirmed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). However, use of AgNO₃ only resulted neither in the formation of NPs nor had any significant effect on the fluorescence of GFP.     

Crystallinity engineering of Au nanoparticles on graphene for in situ SERS monitoring of Fenton-like reaction

Fabrication of multifunctional nanoplatform to in situ monitor Fenton reaction is of vital importance to probe the underlying reaction process and design high-performance catalyst.Herein,a hybrid catalyst comprising of single-crystalline Au nanoparticles（SC Au NPs） on reduced graphene oxide（RGO） sheet was prepared,which not only exhibited an excellent ¹ O₂ mediated Fenton-like catalytic activity in promoting rhodamine 6 G（R6 G） degradation by activating H₂ O_...     

On-chip generation and reaction of unstable intermediates-monolithic nanoreactors for diazonium chemistry: azo dyes

Monolithic nanoreactors for the safe and expedient continuous synthesis of products requiring unstable intermediates were fabricated and tested by the synthesis of azo dyes under hydrodynamic pumping regimes.     

Polymer-based photocoupling agent for the efficient immobilization of nanomaterials and small molecules

A highly efficient photocoupling agent, based on perfluorophenylazide (PFPA)-conjugated polyallylamine (PAAm), was developed for the efficient immobilization of polymers, nanoparticles, graphene, and small molecules. The conjugate, PAAm-PFPA, was synthesized, and the percentage of the photoactive moiety, PFPA, can be controlled by the ratio of the two components in the synthesis. By treating epoxy-functionalized wafers with PAAm-PFPA, photoactive surfaces were generated. Compared with the PFPA surface, these polymer-based photocoupling matrix resulted in significantly enhanced immobilization efficiencies, especially for nanomaterials and small molecules. Thus, polystyrene nanoparticles (PS NPs) and alkyl-functionalized silica nanoparticles (SNPs) were successfully immobilized on the PAAm-PFPA surface, resulting in a high material density. Graphene flakes patterned on the PAAm-PFPA surface showed improved feature resolution in addition to a higher material density compared to that of flakes immobilized on the PFPA surface. Furthermore, 2-O-α-D-mannopyranosyl-D-mannopyranose (Man2) immobilized on the PAAm-PFPA surface exhibited significantly enhanced signals when treated with lectin concanavalin A (Con A).     

Chemocavity: specific concavity in protein reserved for the binding of biologically functional small molecules

The idea that there should be a specific site on a protein for a particular functional small molecule is widespread. It is, however, usually not so easy to understand what characteristics of the site determine the binding ability of the functional small molecule. We have focused on the concurrence rate of the 20 standard amino acids at such binding sites. In order to correlate the concurrence rate and the specific binding site, we have analyzed high-quality X-ray structures of complexes between proteins and small molecules. A novel index characterizing the binding site based on the concurrency rate has been introduced. Using this index we have identified that there is a specific concavity designated as a chemocavity where a specific group of small molecules, i.e., canonical molecular group, is highly inclined to be bound. This study has demonstrated that a chemocavity is reserved for a specific canonical molecular group, and the prevalent idea has been confirmed.     

On-chip fabrication of silver microflower arrays as a catalytic microreactor for allowing in situ SERS monitoring

Silver microflower arrays constructed by upright nanoplates and attached nanoparticles were fabricated inside a microfluidic channel, thus a robust catalytic microreactor for allowing in situ SERS monitoring was proposed. On-chip catalytic reduction shows that the silver microflowers have high catalytic activity and SERS enhancement.     

Highly sensitive in vitro selections for DNA-linked synthetic small molecules with protein binding affinity and specificity

We have developed in vitro selections for DNA-linked synthetic small molecules with protein binding affinity and specificity. These selections require only generally accessible equipment, offer high degrees of enrichment of active molecules from mixtures of predominantly inactive species, can be applied to a variety of unrelated proteins, and require approximately 108-fold less material than existing synthetic molecule screening methods. Iterating these selections multiplies the net enrichment of active molecules, enabling enormous overall enrichment factors exceeding 106 to be achieved. Further, the selections can be adapted to select for binding specificity in addition to binding affinity. The application of methods described in this work may play a key role in the discovery of desired molecules from DNA-templated synthetic libraries.     

Optical sensors with molecularly imprinted nanospheres: a promising approach for robust and label-free detection of small molecules

Molecularly imprinted nanospheres obtained by miniemulsion polymerization have been applied as the sensitive layer for label-free direct optical sensing of small molecules. Using these particles as the sensitive layer allowed for improving response times in comparison to sensors using MIP layers. As a model compound, well-characterized nanospheres imprinted against l-Boc-phenylalanine anilide (l-BFA) were chosen. For immobilization, a simple concept based on electrostatic adsorption was used, showing its applicability to different types of surfaces, leading to a good surface coverage. The sensor showed short response times, good selectivity, and high reversibility with a limit of detection down to 60 μM and a limit of quantitation of 94 μM. Furthermore, reproducibility, selectivity, and long-term stability of the sensitive layers were tested. The best results were achieved with an adsorption on aminopropylsilane layers, showing a chip-to-chip reproducibility of 22%. Furthermore, the sensors showed no loss in signal after a storage time of 1 year.     

Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nAChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole-AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.     

Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification

In vivo carrier protein tagging has recently become an attractive target for the site-specific modification of fusion systems and new approaches to natural product proteomics. A detailed study of pantetheine analogues was performed in order to identify suitable partners for covalent protein labeling inside living cells. A rapid synthesis of pantothenamide analogues was developed and used to produce a panel which was evaluated for in vitro and in vivo protein labeling. Kinetic comparisons allowed the construction of a structure-activity relationship to pinpoint the linker, dye, and bioorthogonal reporter of choice for carrier protein labeling. Finally bioorthogonal pantetheine analogues were shown to target carrier proteins with high specificity in vivo and undergo chemoselective ligation to reporters in crude cell lysate. The methods demonstrated here allow carrier proteins to be visualized and isolated for the first time without the need for antibody techniques and set the stage for the future use of carrier protein fusions in chemical biology.     

In situ formation of dispersed palladium nanoparticles in microemulsion: efficient reaction system for ligand-free Heck reaction

The ligand-free Heck reaction catalyzed by Pd(OAc)2 performed well in a TX10 oil-in-water microemulsion. TEM proved in situ formation distributed palladium nanoparticles in the microemulsion. The role of TX10 in the reaction system is the palladium nanoparticles reducing agent and stabilizer. The effect of reaction parameters on the Heck reaction conversion were discussed. The results indicated that the aqueous phase concentration, the base concentration, and the temperature played key roles in the conversion of the reaction. Iodobenzene was converted to the corresponding trans-stilbene quantitatively within 90-150 min. Therefore, the heptane/TX 10/butanol/water/propylene glycol microemulsion containing in situ formed palladium nanoparticles was a very efficient catalyst system for the ligand-free Heck reaction.     

On-chip Raman analysis of heterogeneous catalytic reaction in supercritical CO2: phase behaviour monitoring and activity profiling

Raman spectroscopy on a chip based Si/glass microreactor allows fast and simultaneous analysis of concentration profiles and phase behaviour of a heterogeneously catalyzed reaction at high pressure.     

Dispersibility of clay and crystallization kinetics for in situ polymerized PET/pristine and modified montmorillonite nanocomposites

Nanocomposite materials composed of poly (ethylene terephthalate) (PET) and montmorillonite (MMT) clays were prepared by in situ polymerization. Samples consisted of PET blended with various quantities of either pristine (Na⁺‐MMT) or organically modified MMT (A10‐MMT). The morphology and thermal and mechanical properties were evaluated for each sample. TEM micrographs, acquired at a 20 nm resolution, provide direct evidence of exfoliation of the clay particles into the PET matrix and show the effect of the alkyl‐modifier on clay dispersibility. The dispersion of PET/A10‐MMT was greater than that observed for the PET/Na⁺‐MMT nanocomposites. The greatest degree of exfoliation occurred for PET/A10‐MMT 0.5 wt %. However, PET/Na⁺‐MMT exhibited higher crystallization temperatures and rates suggesting that Na⁺‐MMT is a more efficient nucleating agent. Both mechanically and thermally, PET/A10‐MMT nanocomposites exhibited superior properties over pure PET. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1022–1035, 2008     

Water PMF for predicting the properties of water molecules in protein binding site

Water is an important component in living systems and deserves better understanding in chemistry and biology. However, due to the difficulty of investigating the water functions in protein structures, it is usually ignored in computational modeling, especially in the field of computer‐aided drug design. Here, using the potential of mean forces (PMFs) approach, we constructed a water PMF (wPMF) based on 3946 non‐redundant high resolution crystal structures. The extracted wPMF potential was first used to investigate the structure pattern of water and analyze the residue hydrophilicity. Then, the relationship between wPMF score and the B factor value of crystal waters was studied. It was found that wPMF agrees well with some previously reported experimental observations. In addition, the wPMF score was also tested in parallel with 3D‐RISM to measure the ability of retrieving experimentally observed waters, and showed comparable performance but with much less computational cost. In the end, we proposed a grid‐based clustering scheme together with a distance weighted wPMF score to further extend wPMF to predict the potential hydration sites of protein structure. From the test, this approach can predict the hydration site at the accuracy about 80% when the calculated score lower than ?4.0. It also allows the assessment of whether or not a given water molecule should be targeted for displacement in ligand design. Overall, the wPMF presented here provides an optional solution to many water related computational modeling problems, some of which can be highly valuable as part of a rational drug design strategy. © 2012 Wiley Periodicals, Inc.     

Deuterium NMR spectroscopy is a versatile and economical tool for monitoring reaction kinetics in ionic liquids

Time-resolved 2H NMR spectroscopy is used to monitor the progress of and gain kinetic information for a variety of reactions in different ionic media.     

In situ prepared CuI nanoparticles on modified poly(styrene-co-maleic anhydride): an efficient and recyclable catalyst for the azide–alkyne click reaction in water

A facile, high-yielding and straightforward methodology for the copper-catalyzed synthesis of 1,4-disubstituted 1,2,3-triazoles in water, using in situ prepared copper nanoparticles (NPs) on modified poly(styrene-co-maleic anhydride) [SMA] catalyst, is reported. The polymer support was easily prepared from the reaction of SMA with 4-aminopyridine and subsequently underwent reaction with CuI NPs. The catalyst was applied for the preparation of triazoles under air, followed by chromatographic separation of the products. The polymer-supported catalyst not only showed high catalytic activity but also showed high 1,4-regioselectivity for the [3 + 2] Huisgen cycloaddition in water as solvent. The products were obtained in good to excellent yields in all cases. The catalyst can be used without pre-activation and reloaded for at least five runs without significant decrease in its activity. The catalyst was characterized by SEM, energy dispersive spectroscopy analysis of X-rays, and inductively coupled plasma.     

Facile synthesis of molybdenum carbide nanoparticles in situ decorated on nitrogen-doped porous carbons for hydrogen evolution reaction

Molybdenum-based electrocatalysts are promising candidates of platinum(Pt)-based materials in electrocatalyzing hydrogen evolution reaction(HER), due to their cost-efficient and resembled electronic properties. Reported herein is the preparation of molybdenum carbide nanoparticles uniformly decorated on nitrogen-modified carbons(Mo_2C/NC) through the carbonization of Mo-based polymers under hydrogen atmosphere by using poly(p-phenylenediamine) and ammonium heptamolybdate polymer analogue as precursors. And the molybdenum nitride nanoparticles loaded on porous N-doped carbons(Mo_2N/NC)are also fabricated by calcination the polymer precursors in nitrogen gas. The Mo_2C/NC shows more excellent electrocatalytic activity than Mo_2N/NC in 0.5 M H_2SO_4, together with robust long-term durability. The well-crystalline nanoparticles and the increased electron conductivity are the main characters responded for the high catalytic efficiency of the fabricated electrocatalysts. This easily fabrication procedure may provide a facile route to prepare non-noble metal carbide/nitride catalysts featuring wellengineered structural and textural peculiarities for realistic energy conversion system.     

The CK2 alpha/CK2 beta interface of human protein kinase CK2 harbors a binding pocket for small molecules

The Ser/Thr kinase CK2 (previously called casein kinase 2) is composed of two catalytic chains (CK2 alpha) attached to a dimer of noncatalytic subunits (CK2 beta). CK2 is involved in suppression of apoptosis, cell survival, and tumorigenesis. To investigate these activities and possibly affect them, selective CK2 inhibitors are required. An often-used CK2 inhibitor is 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In a complex structure with human CK2 alpha, DRB binds to the canonical ATP cleft, but additionally it occupies an allosteric site that can be alternatively filled by glycerol. Inhibition kinetic studies corroborate the dual binding mode of the inhibitor. Structural comparisons reveal a surprising conformational plasticity of human CK2 alpha around both DRB binding sites. After local rearrangement, the allosteric site serves as a CK2 beta interface. This opens the potential to construct molecules interfering with the CK2 alpha/CK2 beta interaction.