Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Rocco, Giulia Di; Bernini, Fabrizio; Borsari, Marco; Martinelli, Ilaria; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Ranieri, Antonio; Caselli, Monica; Sola, Marcó; Ponterini, Glauco

doi:10.1515/zpch-2015-0749

Cited by 4 publications

(2 citation statements)

References 31 publications

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“…To perform an in vitro FRET study of the PDE5C-TC-FlAsH/cGMPS-rhodamine complex, a preliminary characterization of the single emission intensities of the donor (D, FlAsH) and the acceptor (A, rhodamine) was performed. , The kinetics of binding of FlAsH to PDE5C-TC, expressed both in HEK293 and in MLTC-1 Leydig tumor cells, was evaluated. The fluorescence intensity of FlAsH alone was measured over 1 h (Figure A), indicating basal and background emission.…”

Section: Results and Discussionmentioning

confidence: 99%

Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach

et al. 2018

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Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant (K d ) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS− rhodamine conjugate by sildenafil; the K i values were 3.6 ± 0.3 nM (IC 50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC 50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

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Section: Results and Discussionmentioning

confidence: 99%

Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach

et al. 2018

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“…[12][13][14], while recent examples, including metal ions and complexes as ligands, are reported as Refs. [15][16][17][18][19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Intrinsic Fluorometric Reporters of Pteridine Reductase 1, a Target for Antiparasitic Agents

2022

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The intrinsic steady-state and time-resolved fluorescence of Leishmania major pteridine reductase 1, a tetrameric protein target for anti-infective agents, is investigated and deciphered in terms of the contributions from populations of the two tryptophans included in each protein monomer. Signals from these local fluorometric reporters contain molecular-level information on the conformational landscape of this protein and on its interaction with a nanomolar pteridinic inhibitor.

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Met80 and Tyr67 affect the chemical unfolding of yeast cytochromec: comparing the solutionvs.immobilized state

et al. 2020

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Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Cited by 4 publications

References 31 publications

Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach

Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach

Intrinsic Fluorometric Reporters of Pteridine Reductase 1, a Target for Antiparasitic Agents

Met80 and Tyr67 affect the chemical unfolding of yeast cytochromec: comparing the solutionvs.immobilized state

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