Fr Francium

The TetR aptamer induces TetR controlled gene expression, and represents an interesting tool for application in synthetic biology. We have analysed the mechanistic basis for RNA aptamer-based induction of TetR. The aptamer binds TetR with a high affinity in the order of 10⁷ M⁻¹, which is similar to operator DNA binding under the used ionic conditions. We identified the binding epitope of the aptamer on TetR, which consists of amino acids T27, N47 and K48 of both monomers, using loss-of-function analysis and electrophoretic mobility shift assays. Tetracycline-induced conformational changes of TetR led to reorientation of the DNA reading head. This movement destroys the composite binding epitope for the aptamer and leads to reduced RNA binding by one order of magnitude. The aptamer can actively displace TetR from the operator DNA; this could be the key factor for its activity in vivo.

Head-lining: The TetR aptamer induces TetR controlled gene expression. The binding epitope of the aptamer was identified (blue) and overlaps with the helix-turn-helix motif (yellow). Tetracycline-induced conformational changes of TetR impair aptamer binding.

Aptamers are single-stranded nucleic acids that fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. Aptamers are generated by an iterative process known as in vitro selection, which permits their isolation from pools of random sequences. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers (e.g., polyelectrolyte effect). Histones, eukaryotic proteins that make up the core structure of nucleosomes are attractive targets for exploring the binding properties of aptamers because these proteins have positively charged surfaces that bind DNA through noncovalent sequence-independent interactions. Previous selections by our lab and others have yielded DNA aptamers with high affinity but low specificity to individual histone proteins. Whether this is a general limitation of aptamers is an interesting question with important practical implications in the future development of protein affinity reagents. Here we report the in vitro selection of a DNA aptamer that binds to histone H4 with a K_d of 13 nM and distinguishes other core histone proteins with 100 to 480-fold selectivity, which corresponds to a ΔΔG of up to 3.4 kcal mol⁻¹. This result extends our fundamental understanding of aptamers and their ability to fold into shapes that selectively bind alkaline proteins.

Basic selection: Previous selections have yielded DNA aptamers with high affinity but low specificity to individual basic histone proteins. Here we report the in vitro selection of a DNA aptamer that binds to histone H4 with a K_d of 13 nM and distinguishes other core histone proteins with 100–480-fold selectivity.

Tandem tracker: Here we introduce a method for studying the kinetics of protein–small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions.

Pyranose 2-oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H₂O₂. In this study, the mechanistic role of the conserved residues His548 and Asn593 in P2O was investigated by using site-directed mutagenesis, transient kinetics, and pH-dependence studies. As single mutants of H548 resulted in mixed populations of noncovalently bound and covalently linked FAD, double mutants containing H167A were constructed, in which the covalent histidyl-FAD linkage was removed in addition to having the H548 mutation. Single mutants H548A, H548N, H548S, H548D and double mutants (with H167A) could not be reduced by D-glucose. For the H167A/H548R mutant, the flavin could be reduced by D-glucose with the reduction rate constant about 220 times lower than that of the H167A mutant. The pH-dependence studies of H167A/H548R indicated that the rate constant of flavin reduction increased about 360-fold upon a pH rise corresponding to pK_a>10.1, whereas the reactions of the wild-type and H167A mutant enzymes were pH independent. Therefore, the data suggest that a pK_a value of >10.1 in the mutant enzyme is associated with the Arg548 residue, and that this residue must be unprotonated to efficiently catalyze flavin reduction. The data imply that for the wild-type P2O, the conserved His548 should be unprotonated in the pH range studied. The unprotonated His548 can act as a general base to abstract the 2-hydroxyl proton of D-glucose and initiate hydride transfer from the substrate to the flavin. Studies of the single mutant N593H showed that the flavin reduction rate constant was 114 times lower than that of the wild-type enzyme and was pH independent, while the K_d for D-glucose binding was 19 times greater.

Abstract concept: Pyranose 2-oxidase catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H₂O₂. By using site-directed mutagenesis, transient kinetics, and pH-dependence studies, the unprotonated form of a conserved residue (H548) was identified as a catalytic base to abstract the 2-hydroxyl proton of D-glucose, and thus to initiate hydride transfer from the substrate to the flavin.

Phosphorylation of protein kinases is a central mechanism involved in numerous cellular regulatory circuits, both in prokaryotic and eukaryotic cells. An understanding of the structural and functional consequences of protein phosphorylation is of considerable importance for the design of selective, small-molecule kinase inhibitors. NMR spectroscopy is a central method to support structure-based drug design. Here, we present the NMR assignment of the activated p38α kinase and compare it to the NMR assignment of unphosphorylated p38α. Conformational changes in solution induced by activation can be located to the activation loop, an adjacent loop, and an insert part of the polypeptide chain that is specific for the family of mitogen-activated kinases. Deuterium-exchange experiments additionally revealed differences in exchange behavior for two residues in an alanine-rich helix–loop motif that becomes more flexible upon binding of an ATP analogue and a substrate peptide.

Flexible communicator: We have assigned the activated p38α by liquid-state NMR spectroscopy and compared it to the assignment of inactivated p38α as well as X-ray structures. We observed large structural and dynamic changes in the activation loop and two further loop segments, as well as changes in H/D exchange properties, and we assessed the dynamic changes in a ternary complex mimicking binding of ATP/Mg²⁺ and a substrate peptide to activated p38α.

A light on the tiles: A sensor that fluoresces in the presence of specific nucleic acids was designed and characterized. The sensor uses a molecular beacon probe and three adaptor strands to form a five-stranded assembly, a DX-tile, with a specific analyte. This sensor is a highly selective and affordable tool for the real-time analysis of DNA and RNA.

The aggregation of α-synuclein (αS), which is implicated in the pathology of Parkinson's disease, produces fibrils in which layers of parallel, in-register β-sheet–loop–β-sheets are formed. The effects of sequence variation in the loop-forming region (referred to as the linker region) on αS aggregation have yet to be systematically studied. In the study described here, we created and characterized αS variants containing mutations in the linker regions. Our results indicate that although the physicochemical properties of the linker region, evaluated based on an intrinsic property of a single amino acid, still play a significant role in aggregation, additional factors can also determine aggregation of αS linker mutants. Our analyses suggest that these factors include a pairwise potential for parallel in-register β-sheet formation. A linker variant displaying significantly reduced self-aggregation interfered with αS aggregation by inhibiting the conversion of αS soluble species to αS insoluble fibrils. We anticipate that linker mutations could serve as a novel method of creating αS variants that are aggregationdefective and/or inhibit αS aggregation.

Don't stick together: Varying the linker sequence connecting its β-strand-forming domains affects or even inhibits the aggregation of α-synuclein. Although the physicochemical properties of the linker region (evaluated based on an intrinsic property of a single amino acid) play a significant role in aggregation, an additional factor can also determine the aggregation behavior of αS linker variants.

AMD3100 is a potent and selective antagonist of the CXCR4 receptor; it has been shown to block the route of entry of HIV into host T-cells. This compound and its analogues have since been found to act as haematopoietic stem cell mobilisation agents and, more recently, as anti-cancer agents. Here, we have examined a fluorescent derivative of AMD3100, L¹, which offered the potential to assess the behaviour of AMD3100 at the cell surface by using optical imaging modalities. The binuclear Zn^II, Cu^II and Ni^II complexes of L¹ have also been investigated as these metals have been previously shown to enhance the binding properties of AMD3100. Furthermore, Zn^II and Cu^II are known to enhance and quench, respectively, the fluorescence of similar anthracenyl-based ligands. Whilst L¹ demonstrates an ability to inhibit the binding of the anti-CXCR4 monoclonal antibody 12G5 (IC₅₀=0.25–0.9 μM), the incorporation of an anthracenyl moiety resulted in a significantly reduced affinity for CXCR4 compared to AMD3100 (IC₅₀=10 nM). We observed no significant increase in fluorescence intensity following incubation with murine pre-B cells overexpressing CXCR4 compared to a control cell line. This limits the usefulness of L¹ as a fluorescent imaging probe. Interestingly, the Zn^II complex, which carries an overall +4 charge, revealed marginally higher specificity and reduced toxicity in vitro compared to the free ligand, albeit with reduced affinity for CXCR4 (IC₅₀=1.8-5 μM). We suggest that the incorporation of an anthracenyl group contributes to the lipophilic character of the free ligand, thereby resulting in transport across the plasma membrane. This effect is seemingly diminished when the ligand is complexed to charged metal ions.

A fluorescent derivative of the CXCR4 antagonist, AMD3100, and a series of binuclear transition metal complexes have been evaluated. The fluorescence of the free ligand has been shown to be enhanced and dramatically quenched in the presence of zinc and copper, respectively. Here we examine the ability of the free ligand and corresponding zinc complex to assess cellular CXCR4 expression.

Where do you stop? Three recent publications have described how the oxidation of 5-methylcytosine by Tet dioxygenases does not stop at the 5-hydroxymethylcytosine (5hmC) state, rather further oxidation of 5hmC is involved in DNA demethylation. The nature of the enzymes involved in this process shed light on the dynamics of epigenetic signaling and its evolutionary origin.

The catalytic regio- and stereoselective oxidation of aliphatic CH bonds by using atmospheric dioxygen as the oxidant is a highly desirable reaction. Reetz and co-workers have recently reported the development of P450_BM3 mutants that oxidise testosterone with greatly increased turnover activity, total stereoselectivity and >95 % regioselectivity.