Mapping the role of aromatic amino acids within a blue-light sensing LOV domain

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of the flavin triplet excited state. LOV domains where the reactive cysteine has been mutated are valuable fluorescent tools for microscopy and as genetically encoded photosensitisers for reactive oxygen species. Besides being convenient tools for applications, LOV domains without the reactive cysteine (naturally occurring or engineered) can still be functionally photoactivated via formation of a neutral flavin radical. Tryptophans and tyrosines are held as the main partners as potential electron donors to the flavin excited states. In this work, we explore the relevance of aromatic amino acids in determining the photophysical features of the LOV protein Mr4511 from Methylobacterium radiotolerans by introducing point mutations into the C71S variant that does not form the covalent adduct. By using an array of spectroscopic techniques we measured the fluorescence quantum yields and lifetimes, the triplet yields and lifetimes, and the efficiency of singlet oxygen (SO) formation for eleven Mr4511 variants. Insertion of Trp residues at distances between 0.6 and 1.5 nm from the flavin chromophore results in strong quenching of the flavin excited triplet state and, at the shorter distances even of the singlet excited state. The mutation F130W (ca. 0.6 nm) completely quenches the singlet excited state, preventing triplet formation: in this case, even if the cysteine is present, the photo-adduct is not formed. Tyrosines are also quenchers for the flavin excited states, although not as efficient as Trp residues, as demonstrated with their substitution with the inert phenylalanine. For one of these variants, C71S/Y116F, we found that the quantum yield of formation for singlet oxygen is 0.44 in aqueous aerobic solution, vs 0.17 for C71S. Based on our study with Mr4511 and on literature data for other LOV domains we suggest that Trp and Tyr residues too close to the flavin chromophore (at distances less than 0.9 nm) reduce the yield of photoproduct formation and that introduction of inert Phe residues in key positions can help in developing efficient, LOV-based photosensitisers. This journal i

Formation of targeted monovalent quantum dots by steric exclusion

Precise control over interfacial chemistry between nanoparticles and other materials remains a significant challenge limiting the broad application of nanotechnology in biology. To address this challenge, we use “Steric Exclusion” to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping the QD with a functionalized oligonucleotide. We demonstrate the utility of these QDs as modular and non-perturbing imaging probes by tracking individual Notch receptors on live cells

Dispersion force for materials relevant for micro and nanodevices fabrication

The dispersion (van der Waals and Casimir) force between two semi-spaces are calculated using the Lifshitz theory for different materials relevant for micro and nanodevices fabrication, namely, gold, silicon, gallium arsenide, diamond and two types of diamond-like carbon (DLC), silicon carbide, silicon nitride and silicon dioxide. The calculations were performed using recent experimental optical data available in the literature, usually ranging from the far infrared up to the extreme ultraviolet bands of the electromagnetic spectrum. The results are presented in the form of a correction factor to the Casimir force predicted between perfect conductors, for the separation between the semi-spaces varying from 1 nanometre up to 1 micrometre. The relative importance of the contributions to the dispersion force of the optical properties in different spectral ranges is analyzed. The role of the temperature for semiconductors and insulators is also addressed. The results are meant to be useful for the estimation of the impact of the Casimir and van der Waals forces on the operational parameters of micro and nanodevices

Reactivity-Dependent PCR: Direct, Solution-Phase in Vitro Selection for Bond Formation

In vitro selection is a key component of efforts to discover functional nucleic acids and small molecules from libraries of DNA, RNA, and DNA-encoded small molecules. Such selections have been widely used to evolve RNA and DNA catalysts and, more recently, to discover new reactions from DNA-encoded libraries of potential substrates. While effective, current strategies for selections of bond-forming and bond-cleaving reactivity are generally indirect, require the synthesis of biotin-linked substrates, and involve multiple solution-phase and solid-phase manipulations. In this work we report the successful development and validation of reactivity-dependent PCR (RDPCR), a new method that more directly links bond formation or bond cleavage with the amplification of desired sequences and that obviates the need for solid-phase capture, washing, and elution steps. We show that RDPCR can be used to select for bond formation in the context of reaction discovery and for bond cleavage in the context of protease activity profiling.Chemistry and Chemical Biolog

Interaction-Dependent PCR: Identification of Ligand−Target Pairs from Libraries of Ligands and Libraries of Targets in a Single Solution-Phase Experiment

Interaction-dependent PCR (IDPCR) is a solution-phase method to identify binding partners from combined libraries of small-molecule ligands and targets in a single experiment. Binding between DNA-linked targets and DNA-linked ligands induces formation of an extendable duplex. Extension links codes that identify the ligand and target into one selectively amplifiable DNA molecule. In a model selection, IDPCR resulted in the enrichment of DNA encoding all five known protein−ligand pairs out of 67 599 possible sequences.Chemistry and Chemical Biolog

In Vitro Selection of a DNA-Templated Small-Molecule Library Reveals a Class of Macrocyclic Kinase Inhibitors

DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13 824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC50 values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.Chemistry and Chemical Biolog

Involvement of peripheral ionotropic glutamate receptors in orofacial thermal hyperalgesia in rats

<p>Abstract</p> <p>Background</p> <p>The purpose of the present study was to elucidate the mechanisms that may underlie the sensitization of trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) neurons to heat or cold stimulation of the orofacial region following glutamate (Glu) injection.</p> <p>Results</p> <p>Glu application to the tongue or whisker pad skin caused an enhancement of head-withdrawal reflex and extracellular signal-regulated kinase (ERK) phosphorylation in Vc-C2 neurons. Head-withdrawal reflex and ERK phosphorylation were also enhanced following cold stimulation of the tongue but not whisker pad skin in Glu-injected rats, and the head-withdrawal reflex and ERK phosphorylation were enhanced following heat stimulation of the tongue or whisker pad skin. The enhanced head-withdrawal reflex and ERK phosphorylation after heat stimulation of the tongue or whisker pad skin, and those following cold stimulation of the tongue but not whisker pad skin were suppressed following ionotropic glutamate receptor antagonists administration into the tongue or whisker pad skin. Furthermore, intrathecal administration of MEK1/2 inhibitor PD98059 caused significant suppression of enhanced head-withdrawal reflex in Glu-injected rats, heat head-withdrawal reflex in the rats with Glu injection into the tongue or whisker pad skin and cold head-withdrawal reflex in the rats with Glu injection into the tongue.</p> <p>Conclusions</p> <p>The present findings suggest that peripheral Glu receptor mechanisms may contribute to cold hyperalgesia in the tongue but not in the facial skin, and also contribute to heat hyperalgesia in the tongue and facial skin, and that the mitogen-activated protein kinase cascade in Vc-C2 neurons may be involved in these Glu-evoked hyperalgesic effects.</p

Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

BACKGROUND: Cellulose acetate phthalate (CAP), a promising candidate microbicide for prevention of sexual transmission of the human immunodeficiency virus type 1 (HIV-1) and other sexually transmitted disease (STD) pathogens, was shown to inactivate HIV-1 and to block the coreceptor binding site on the virus envelope glycoprotein gp120. It did not interfere with virus binding to CD4. Since CD4 is the primary cellular receptor for HIV-1, it was of interest to study CAP binding to HIV-1 complexes with soluble CD4 (sCD4) and its consequences, including changes in the conformation of the envelope glycoprotein gp41 within virus particles. METHODS: Enzyme-linked immunosorbent assays (ELISA) were used to study CAP binding to HIV-1-sCD4 complexes and to detect gp41 six-helix bundles accessible on virus particles using antibodies specific for the α-helical core domain of gp41. RESULTS: 1) Pretreatment of HIV-1 with sCD4 augments subsequent binding of CAP; 2) there is synergism between CAP and sCD4 for inhibition of HIV-1 infection; 3) treatment of HIV-1 with CAP induced the formation of gp41 six-helix bundles. CONCLUSIONS: CAP and sCD4 bind to distinct sites on HIV-1 IIIB and BaL virions and their simultaneous binding has profound effects on virus structure and infectivity. The formation of gp41 six-helical bundles, induced by CAP, is known to render the virus incompetent for fusion with target cells thus preventing infection

Validation of the SCID-hu Thy/Liv mouse model with four classes of licensed antiretrovirals.

BackgroundThe SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals.Methodology/principal findingsEndpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-resistance substitutions.ConclusionGiven the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans