Time-Gated Luminescence Detection of Enzymatically Produced Hydrogen Sulfide: Design, Synthesis, and Application of a Lanthanide-Based Probe.

Hydrogen sulfide (H2S) is now recognized as an important gaseous transmitter that is involved in a variety of biological processes. Here, we report the design and synthesis of a luminescent lanthanide biosensor for H2S, LP2-Cu(II)-Ln(III), a heterobinuclear metal complex that uses Cu(II) decomplexation to control millisecond-scale-lifetime-Tb(III)- or Eu(III)-emission intensity. LP2-Cu(II)-Ln(III) responded rapidly, selectively, and with high sensitivity to aqueous H2S. The probe's potential for biological applications was verified by measuring the H2S generated by the slow-releasing chemical-sulfide-donor GYY4147, by cystathionine γ-lyase (CSE), and by Na2S-stimulated HeLa cells.

Ratiometric QD-FRET Sensing of Aqueous H2S in Vitro.

We report a platform for the ratiometric fluorescent sensing of endogenously generated gaseous transmitter H2S in its aqueous form (bisulfide or hydrogen sulfide anion) based on the alteration of Förster resonance energy transfer from an emissive semiconductor quantum dot (QD) donor to a dithiol-linked organic dye acceptor. The disulfide bridge between the two chromophores is cleaved upon exposure to bisulfide, resulting in termination of FRET as the dye diffuses away from the QD. This results in enhanced QD emission and dye quenching. The resulting ratiometric response can be correlated quantitatively to the concentration of bisulfide and was found to have a detection limit as low as 1.36 ± 0.03 µM. The potential for use in biological applications was demonstrated by measuring the response of the QD-based FRET sensor microinjected into live HeLa cells upon extracellular exposure to bisulfide. The methodology used here is built upon a highly multifunctional platform that offers numerous advantages, such as low detection limit, enhanced photochemical stability, and sensing ability within a biological milieu.

Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2.

Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at non-cognate ligand binding sites to alter the allosteric activation process are classified as allosteric modulators and represent a promising class of therapeutics with distinct modes of binding and action. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed fluorescence resonance energy transfer (FRET) sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM (egative allosteric modulator) increases the occupancy of one of the intermediate states while a positive allosteric modulator increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.