Fr Francium

We report the sensitive detection of telomerase activity by using exonuclease III-aided target recycling to amplify the signal produced by a chimeric LNA–DNA molecular beacon. We demonstrate the specific detection of as few as 30 telomerase-positive breast cancer cells in a single-measurement fluorescence assay that avoids the problematic PCR and gel analysis of the current “gold-standard” assay.

LynF prenylates, but the prenyl migrates: Schmidt and co-workers have demonstrated that LynF from Lyngbya aestuarii is a reverse O-prenyl transferase. However, a forward C-prenylated product is obtained through a non-enzymatic Claisen rearrangement. The elucidation of this unprecedented two-step process is a significant contribution to our understanding of the biosynthesis of complex macrocyclic peptides.

Triple-helix-forming oligonucleotides (TFOs) are widespread in the genome and have been found in regulatory regions, especially in promoter zones and recombination hotspots of DNA. To specifically detect these polypurine sequences, we designed and synthesized two dual pyrene-labeled single-strand oligonucleotide probes (TFO-FPs) consisting of recognition, linker, and detection sequences. The hybridization processes of TFO-FPs with target polypurine oligonucleotides involve both Watson–Crick and Hoogsteen base-pairings. Through double sensing of oligonucleotide sequences, single mutations of target oligonucleotides are detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrifice of hybridization kinetics.

A double sensing strategy for detecting mismatching in polypurine oligonucleotides has been developed. Double sensing by the oligonucleotide sequence allows single mutations of target oligonucleotides to be detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrificing hybridization kinetics.

Rab GTPases play a key role in the regulation of membrane trafficking. Post-translational geranylgeranylation is critical for their biological activity and is conferred by Rab geranylgeranyl transferease (RabGGTase), together with an accessory factor, Rab escort protein (REP). Mechanistic studies of Rab prenylation and identification of RabGGTase inhibitors require sensitive reporters of Rab prenylation. In the present work, a combination of protein engineering and expressed protein ligation was used to construct a library of semisynthetic Rab7 fluorescent conjugates. In order to avoid synthesis of a large number of fluorescently labeled peptides, we developed a strategy that combined thiol-reactive dye-labeling of cysteine with in vitro protein ligation. Application of this strategy required optimization of labeling and ligation conditions to promote thiol labeling and disfavor intramolecular cyclization. Using this approach, we constructed 46 fluorescent sensors with different spectral properties that reported on the interaction of Rab7 with RabGGTase, REP-1, and the overall prenylation reaction. Two constructs, Rab7Δ3CCK(NBD) and Rab7Δ2SCCC–dans, displayed 2.5- and 1.5-fold increase in fluorescence, respectively, upon prenylation. Moreover, dansyl-, NBD (4-nitro-benzofurazan)-, I-BA-, and I-SO-labeled Rab7 conjugates exhibited two- to tenfold change in fluorescence upon binding to REP or RabGGTase. These fluorescent sensors allowed us to monitor Rab prenylation in real time and to investigate the assembly of Rab–REP binary and Rab–REP–RabGGTase ternary complexes.

Taking the Rab: Expressed protein ligation allows site-specific incorporation of unnatural functionalities, such as fluorophores, into proteins. This requires synthesis of peptides with such functionalities and their subsequent ligation onto proteins. To construct a library of fluorescent RabGTPase sensors we developed an alternative approach in which proteins are labeled on a free cysteine and then ligated to an unlabeled peptide.

The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.

Additional epoxidation: A two-component flavin-dependent monooxygenase, the ActVA-ORF5/ActVB system, functioning as a quinone-forming C-6 oxygenase for actinorhodin biosynthesis in Streptomyces coelicolor, was revealed to have an additional epoxidation activity in vitro. This additional function is possibly a result of the adaptable structural features of the two-component system.

Conformationally constrained peptidomimetics could be of great value in the design of vaccines targeting protective epitopes on viral and bacterial pathogens. But the poor immunogenicity of small synthetic molecules represents a serious obstacle for their use in vaccine development. Here, we show how a constrained epitope mimetic can be rendered highly immunogenic through multivalent display on the surface of synthetic virus-like nanoparticles. The target epitope is the V3 loop from the gp120 glycoprotein of HIV-1 bound to the neutralizing antibody F425-B4e8. The antibody-bound V3 loop adopts a β-hairpin conformation, which is effectively stabilized by transplantation onto a D-Pro-L-Pro template. The resulting mimetic after coupling to synthetic virus-like particles elicited antibodies in rabbits that recognized recombinant gp120. The elicited antibodies also blocked infection by the neutralization sensitive tier-1 strain MN of HIV-1, as well as engineered viruses with the V1V2 loop deleted; this result is consistent with screening of V3 by the V1V2 loop in intact trimeric viral gp120 spikes. The results provide new insights into HIV-1 vaccine design based on the V3 loop, and illustrate how knowledge from structural biology can be exploited for the design of constrained epitope mimetics, which can be delivered to the immune system by using a highly immunogenic synthetic nanoparticle delivery system.

Display article: A structural vaccinology approach could lead to a new generation of vaccine candidates, based upon conformationally constrained epitope mimetics. Here a β-hairpin mimetic of the HIV-1V3 loop is rendered immunogenic by multivalent display on the surface of synthetic virus-like particles (SVLPs).

We present a new probe for the determination of hydrogen peroxide (HP). It is based on the yellow fluorophore 4-amino-1,8-napththalimide, coupled to p-anisidine (as a redox-active group) to form a probe that is based on photoinduced electron transfer (PET). The preparation of the probe (which we refer to as “HP Green”) was accomplished in four steps with good yield. Its fluorescence is independent of pH in the physiological range and quenched by a PET process that occurs between the p-anisidine redox moiety and the naphthalimide luminophore. If the p-anisidine group is oxidized by HP, PET is suppressed and fluorescence intensity is strongly increased. Addition of horseradish peroxidase (HRP) enhances the oxidation of HP Green and further improves the detection limit of HP. The use of HRP and HP Green enables the determination of HP concentration in a range of 0.1 to 5 μM, with a limit of detection (LOD) as low as 64 nM (16 pmol per well in microtiter plates). HP Green and HRP also enable sensitive enzymatic assays of oxidase substrates in a kinetic format, as shown for L-lactate and D-glucose. L-Lactate concentration can be rapidly determined between 0.5 and 10 μM after 6 minutes of incubation at 30 °C, with an LOD of 164 nm (41 pmol per well). This LOD is more than sixfold lower than that of the best commercial assays for lactate. The detection range for D-glucose is 2 to 30 μm, and the LOD is 644 nM (161 pmol per well). These are among the lowest concentrations detectable for oxidase-based assays. The hexanoic acid moiety in HP Green may be further used to immobilize the probe in order to obtain sensor layers for continuous assays.

Suppression of the photoinduced electron transfer (PET) in a new fluorescent probe (HP Green) for hydrogen peroxide uses a naphthalimide fluorophore coupled to p-anisidine (as a redoxactive group). It has been shown to enable rapid and highly sensitive enzymatic assays for L-lactate (limit of detection, LOD, 162 nM) and D-glucose (LOD 0.64 μM).

Control yourself! A two-color molecular beacon with non-nucleosidic chromophores in a triplex stem is presented. Pyrene and PDI fluorophores act as mutual quenchers by formation of a donor–acceptor complex in the closed form. Hybridization with the target results in two independent fluorescence signals. The two-color read-out provides a “self-control” feature, which helps to eliminate false positive signals in imaging and screening applications.

This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent-driven fusion of large vesicles (0.1–0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein-reconstituted large unilamellar vesicles (LUVs) with a lipid-containing solvent phase. We made GPVs by using n-decane and squalene as solvents, and applied generalized polarization (GP) imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity-sensitive probe Badan. Specifically, we created GPVs of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform.

Polarity exploration: A method to create giant protein vesicles (GPVs) is described. Fluorescence microscopy is used to characterize GPV morphology and protein–lipid hydrophobic interactions. Specifically, we employed generalized polarization imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity-sensitive probe Badan.

Open and closed: The characterization of protein mobility is crucial for the understanding of biological functions. We have applied NMR spectroscopy to study the conformational dynamics of the 80 kDa enzyme prolyl oligopeptidase (POP). Our results revealed that POP is highly dynamic and that inhibition of catalytic activity shifts this conformational equilibrium towards a less dynamic state.