Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development.     

Pharmacophore-based strategy for the development of general and specific scFv biosensors for abused antibiotics

We developed fluorescent biosensor systems that are either general or selective to fluoroquinolone antibiotics by using a single-chain variable-fragment (scFv) as a recognition element. The selectivity of these biosensors to fluoroquinolone antibiotics was rationally tuned through the structural modification on the pharmacophore of fluoroquinolone antibiotics and the subsequent selection of scFv receptor modules against these antibiotics-based antigens using phage display. The resulting A2 and F9 scFv's bound to their representative antigen with a moderate affinity (K(D) in micromolar range as determined by surface plasmon resonance). A2 is a specific binder for enrofloxacin and did not cross-react with other fluoroquinolone antibiotics including structurally similar ciprofloxacin, while F9 is a general fluoroquinolone binder that likely bound to the antigen at the common pyridone-carboxylic acid pharmacophore. These scFv-based receptors were successfully applied to the development of one-step fluorescent biosensor which can detect fluoroquinolone antibiotics at concentrations below the level suggested in animal drug application guidelines. The strategy described in this report can be applied to developing convenient field biosensors that can qualitatively detect overused/misused antibiotics in the livestock drinking water.     

Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase,an Initial Component of the N-End Rule Pathway

The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.     

Bioconjugation of L-3,4-dihydroxyphenylalanine containing protein with a polysaccharide

We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-l-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications.     

Overexpression of DRG2 suppresses the growth of Jurkat T cells but does not induce apoptosis

Developmentally regulated GTP-binding protein (DRG) is a new subfamily within the superfamily of GTP-binding proteins. Its expression is regulated during embryonic development. To investigate the effect of the expression of DRG2 on cell growth, we constructed a human Jurkat-T-cell line that overexpresses DRG2. Overexpression of DRG2 suppressed the growth and the aggregation of Jurkat cells but did not induce apoptotic cell death. We used cDNA microarray analysis to examine the global changes in gene expression induced by an overexpression of DRG2. DNA array analyses identified genes that may suppress cell growth at a number of levels in multiple signaling cascades in Jurkat cells and also several prosurvival genes that may protect cells from apoptosis.     

Effect of a polyoxydonium immunoregulator on the biological properties of microorganisms

The effect of the synthetic immunomodulator polyoxydonium (PO) on some biological properties of pathogenic bacteria (Shigella flexneri, Salmonella enteritidis), opportunistic bacteria (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacteroides fragilis, Peptostreptococcus anaerobius, Prevotella melaninogenica, Propionibacterium propionicum, Clostridium difficile) and fungi (Candida albicans), isolated during enteric infections, enteric dysbiosis, pyoinflammatory diseases, was evaluated in a number of in vitro experiments. The study revealed that the recommended therapeutic concentration of PO decreased antilysozyme activity (ALA) and the anticomplement activity in Klebsiella, Shigella, Propionibacterium, Clostridium, bacteroids, fungi of the genus Candida, but increased ALA in nonhemolytic Escherichia. Under the action of PO an increased sensitivity of the microorganisms under study to definite antibiotics of the lincosamide, fluoroquinolon, carbapenem and cephalosporin groups was noted. The data obtained in this study reveal one of the possible mechanisms of the corrective action of PO on the microbiocenosis of the intestine in dysbiosis, enteric infections and pyoinflammatory diseases.