A semisynthetic fluorescent sensor protein for glutamate

We report the semisynthesis of a fluorescent glutamate sensor protein on cell surfaces. Sensor excitation at 547 nm yields a glutamate-dependent emission spectrum between 550 and 700 nm that can be exploited for ratiometric sensing. On cells, the sensor displays a ratiometric change of 1.56. The high sensitivity toward glutamate concentration changes of the sensor and its exclusive extracellular localization make it an attractive tool for glutamate sensing in neurobiology.     

Structural chemistry of a green fluorescent protein Zn biosensor

We designed a green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) (enhancing fluorescence intensity) and Cu(II) (quenching fluorescence) directly to a chromophore ligand that resembles a dipyrrole unit of a porphyrin. Crystallographic structure determination of apo, Zn(II)-bound, and Cu(II)-bound BFPms1 to better than 1.5 A resolution allowed us to refine metal centers without geometric restraints, to calculate experimental standard uncertainty errors for bond lengths and angles, and to model thermal displacement parameters anisotropically. The BFPms1 Zn(II) site (KD = 50 muM) displays distorted trigonal bipyrimidal geometry, with Zn(II) binding to Glu222, to a water molecule, and tridentate to the chromophore ligand. In contrast, the BFPms1 Cu(II) site (KD = 24 muM) exhibits square planar geometry similar to metalated porphyrins, with Cu(II) binding to the chromophore chelate and Glu222. The apo structure reveals a large electropositive region near the designed metal insertion channel, suggesting a basis for the measured metal cation binding kinetics. The preorganized tridentate ligand is accommodated in both coordination geometries by a 0.4 A difference between the Zn and Cu positions and by distinct rearrangements of Glu222. The highly accurate metal ligand bond lengths reveal different protonation states for the same oxygen bound to Zn vs Cu, with implications for the observed metal ion specificity. Crystallographic anisotropic thermal factor analysis validates metal ion rigidification of the chromophore in enhancement of fluorescence intensity upon Zn(II) binding. Thus, our high-resolution structures reveal how structure-based design has effectively linked selective metal binding to changes in fluorescent properties. Furthermore, this protein Zn(II) biosensor provides a prototype suitable for further optimization by directed evolution to generate metalloprotein variants with desirable physical or biochemical properties.     

人工细胞膜上天然酶与人工受体的分子间通讯

在集成的分子系统中,分子间的通讯联系是设计分子器件和分子机械的重要方式[1].在水相介质中自组装形成的脂质体可以作为分子间通讯的平台,各种分子可以依靠弱作用力,按照设计思路有组织、有计划地排布其上,构成一个功能化的超分子体,即纳米器件(nanodevice).在脂质体上模拟生物膜上发生的细胞信号转导,成为在分子和超分子水平上开发具有仿生特征的新型纳米器件的重要方法和手段,近年来成为关注的热点[2-6].     

Rapid protein expression analysis with an interferometric biosensor for monitoring protein production

The concentration of a recombinantly expressed protein has to be monitored to select optimal expression conditions throughout the protein production process. Today this is usually achieved semiquantitatively with sodium dodecyl sulfate polyacrylamide gel electrophoresis/western blotting or with ELISAs, which are time- and labor-intensive methods. In this paper the applicability of a label-free sensor system based on a Young interferometer is presented as an alternative for the monitoring of recombinant protein production. Once a protein is successfully produced, the interferometric biosensor allows any protein–protein interaction to be characterized in a label-free manner. This is demonstrated with an antibody/antigen pair, where the antibody is directed against a four-amino-acid tag used for protein expression analysis as well as purification during recombinant protein production. Label-free detection of the tagged protein is shown both in buffer and in bacterial cell lysate as a sample matrix. The system exhibiting a low limit of detection, low drift and reliable operation is compared with a commercial surface plasmon resonance sensor and a competitive ELISA. Figure 1 Waveguide sensor chip; grating (green) illuminated by a red light source. Image courtesy of Unaxis Optics Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Tetraethylorthosilicate film modified with protein to fabricate cholesterol biosensor

Sol-gel derived tetraethylorthosilicate (TEOS) films were prepared by spin coating method on indium tin oxide (ITO) coated glass plate. Hydrophobic interaction method was used to coat the bovine serum albumin film over the surface of tetraethylorthosilicate sol-gel film to minimize cracking, biofouling and to improve the stability of the film. Cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) were immobilized using covalent linkage with bovine albumin serum film to enhance the loading of the enzyme to improve the sensitivity of biosensor. Further ITO-TEOS-BSA-ChOx/HRP film was characterized by UV-vis, FTIR and SEM techniques. The optical response of the ITO-TEOS-BSA-ChOx/HRP biosensor was found to be linear in the range of 2-8 mM for cholesterol concentration with response time approximately 20 s. Amperometric response of ITO-TEOS-BSA-ChOx/HRP was observed to be linear in the range of 2-12 mM of cholesterol concentration with 10-s response time. Michaelis-Menten constant was calculated 21.2 mM .The shelf life of ITO-TEOS-BSA-ChOx/HRP biosensor was approximately about 8 weeks in desiccated conditions at room temperature.     

Coupling artificial actin cortices to biofunctionalized lipid monolayers

We report the assembly of protein supramolecular structures at an air-water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin-biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.     

Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence.The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised.The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.     

Electrochemical uranyl biosensor with DNA oligonucleotides as receptor layer

The feasibility of using gold electrodes modified with short-chain ssDNA oligonucleotides for determination of uranyl cation is examined. Interaction between UO₂²⁺ and proposed recognition layer was studied by means of voltammetric and quartz crystal microbalance measurements. It was postulated that ssDNA recognition layer functions via strong binding of UO₂²⁺ to phosphate DNA backbone. The methylene blue was used as a redox marker for analytical signal generation. Biosensor response was based on the difference in electrochemical signal before and after subjecting it to sample containing uranyl ion. The lower detection limit of 30 nmol L⁻¹ for UO₂²⁺ was observed for a sample incubation time of 60 min. Proposed ssDNA-modified electrodes demonstrated good selectivity towards UO₂²⁺ against common metal cations, with only Pb²⁺ and Ca²⁺ showing considerable interfering effect.     

Solid-state electrochemiluminescence protein biosensor with aptamer substitution strategy

One solid-state electrochemiluminescence(ECL) protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed.Additionally,the biosensor was based on ECL photo-quenching effect of ferrocene(Fc) to tris(2,2'-bipyridyl)ruthenium(II)(Ru(bpy)32+).It was built up by modification of Au nanoparticles(AuNPs) and Ru(bpy)3 2+ on one Au electrode firstly,and then self-assembly of one special double-stranded DNA(dsDNA) onto the electrode.This ...     

A new fluorescent chemosensor for anion based on an artificial cyclic tetrapeptide

A cyclic tetrapeptide composed of alternating glycine and 8-amino-4-iso-butoxyquinoline-2-carboxylic acid was designed and synthesized. Its complexation properties with anions were performed by fluorescence spectroscopy and ¹H NMR method.     

Design of a hybrid biosensor for enhanced phosphopeptide recognition based on a phosphoprotein binding domain coupled with a fluorescent chemosensor

Protein-based fluorescent biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated biological systems. Here, we present the design of a hybrid biosensor, specific for a bis-phosphorylated peptide, based on a natural phosphoprotein binding domain coupled with an artificial fluorescent chemosensor. The hybrid biosensor consists of a phosphoprotein binding domain, the WW domain, into which has been introduced a fluorescent stilbazole having Zn(II)-dipicolylamine (Dpa) as a phosphate binding motif. It showed strong binding affinity and high sensing selectivity toward a specific bis-phosphorylated peptide in the presence of various phosphate species such as the monophosphorylated peptide, ATP, and others. Detailed fluorescence titration experiments clearly indicate that the binding-induced fluorescence enhancement and the sensing selectivity were achieved by the cooperative action of both binding sites of the hybrid biosensor, i.e., the WW domain and the Zn(II)-Dpa chemosensor unit. Thus, it is clear that the tethered Zn(II)-Dpa-stilbazole unit operated not only as a fluorescence signal transducer, but also as a sub-binding site in the hybrid biosensor. Taking advantage of its selective sensing property, the hybrid biosensor was successfully applied to real-time and label-free fluorescence monitoring of a protein kinase-catalyzed phosphorylation.     

Chromogenic and fluorescent recognition of iodide with a benzimidazole-based tripodal receptor

A novel 1,3,5-substituted triethylbenzene derivative with a 2-aminobenzimidazole moiety as a binding and signaling subunit was synthesized. The sensor was tested in a buffered CH(3)CN/H(2)O (99:1, v/v) solution and found to be selective for iodide as demonstrated by the photophysical properties obtained through UV-vis absorption and fluorescence spectroscopy analyses.     

Construction of a small-molecule-integrated semisynthetic split intein for in vivo protein ligation

A new split intein-based protein ligation tool that is synthetically accessible and can be used for protein semisynthesis on the cell surface and potentially inside cells has been constructed.     

Rational design of a novel fluorescent biosensor for beta-lactam antibiotics from a class A beta-lactamase

A rational design strategy was used to construct a sensitive "turn-on" biosensor for beta-lactam antibiotics and beta-lactamase inhibitors from a class A beta-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Omega-loop of the E166C mutant close to the active site of the beta-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Omega-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent. This process is accompanied by an increase in the fluorescence of the labeled enzyme. The fluorescence intensity of the biosensor increases with the concentration of antibiotics or inhibitors, which can detect penicillin G at concentrations as low as 50 nM in water. This approach opens a possibility for converting highly active and nonallosteric enzymes into substrate-binding proteins for biosensing purposes.     

Highly sensitive cyanide anion detection with a coumarin-spiropyran conjugate as a fluorescent receptor

A coumarin-spiropyran conjugate (2) shows a CN(-)-selective fluorescence enhancement under UV irradiation. This enables accurate determination of very low levels of CN(-) (>0.5 μM).     

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein

We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.     

Ratiometric and water-soluble fluorescent zinc sensor of carboxamidoquinoline with an alkoxyethylamino chain as receptor

A novel "naked-eye" and ratiometric fluorescent zinc sensor (AQZ) of carboxamidoquinoline with an alkoxyethylamino chain as receptor was designed and synthesized. AQZ shows good water solubility and high selectivity for sensing; about an 8-fold increase in fluorescence quantum yield and a 75 nm red-shift of fluorescence emission upon binding Zn2+ in buffer aqueous solution are observed. Moreover, AQZ can enter yeast cells and signal the presence of Zn2+.     

Fluorophores related to the green fluorescent protein

Imidazolin-5-one derivatives and isosteres (oxazolinones, butenolides, and pyrrolinones) of the 4-hydroxybenzylidene-imidazolinone chromophore of the GFP have been synthesized and their photophysical properties have been investigated.