Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy

Fernandes, Dennis D.; Bamrah, Jasbir; Kailasam, Senthilkumar; Gomes, Gregory-Neal W.; Li, Yuchong; Wieden, Hans-Joachim; Gradinaru, Claudiu C.

doi:10.1038/s41598-017-13427-8

Cited by 11 publications

(14 citation statements)

References 68 publications

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“…Observed changes in the FRET efficiency reflect displacements of TM6 relative to TM4, which remains relatively immobile (Supporting Information Section 1, Figure S1). Solution smFRET experiments on detergent-reconstituted A2AR samples were performed on a custom-built single-molecule fluorescence microscope and the multiparameter photon data was analyzed using custom MATLAB scripts [35][36] . Labelled protein was diluted to 50 pM in a buffer solution (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% MNG-3, 0.02% CHS, 10 mM cysteamine) to ensure a low probability (< 1%) of two independent receptors simultaneously occupying the confocal detection volume.…”

Section: Resultsmentioning

confidence: 99%

“…Single-Molecule FRET. smFRET measurements were performed on a custom-built multiparameter fluorescence microscope with alternating-laser excitation (ALEX) [35][36] . The donor dye (AF488) was excited with a 473 nm diode-pumped solid-state laser (Cobolt Blue, Market Tech).…”

Section: Methodsmentioning

confidence: 99%

“…where N is the total number of photon counts detected and Bi is the fraction of species with the lifetime τLi 36 .…”

Section: Methodsmentioning

confidence: 99%

“…decay. Fluorescence lifetime and anisotropy decay measurements were performed using the custom-built multiparameter fluorescence microscope (see above) and the data was fit using custom MATLAB code36 . BODIPY-FL was excited with 480-nm femtosecond pulses obtained by frequency-doubling the output of a tunable Ti:Sapphire laser (Tsunami HP, Spectra Physics, USA).…”

mentioning

confidence: 99%

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Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

Fernandes

Neale

Gomes

et al. 2020

Preprint

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G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra-and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 μs) and slow (200-300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…where N is the total number of photon counts detected and Bi is the fraction of species with the lifetime τLi 36 .…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

Fernandes

Neale

Gomes

et al. 2020

Preprint

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“…Kappa 2 calculation. The dipole orientation factor, κ 2 , was calculated using an approach as described previously 39…”

Section: Methodsmentioning

confidence: 99%

The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

Pope

Johnson

Jamieson

et al. 2019

Preprint

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Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying ~86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.

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Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer

et al. 2020

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Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio-orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild-type FPs are fused to a naturally occurring protein-protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.

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Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy

Cited by 11 publications

References 68 publications

Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer

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Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy

Cited by 11 publications

References 68 publications

Ligand Modulation of the Conformational Dynamics of the A2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

Ligand Modulation of the Conformational Dynamics of the A2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer

Contact Info

Product

Resources

About

Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

Ligand Modulation of the Conformational Dynamics of the A_2AAdenosine Receptor Revealed by Single-Molecule Fluorescence