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Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore

[Image: see text] We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently...

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Autores principales: Lee, Molly M., Peterson, Blake R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2016
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204206/
https://www.ncbi.nlm.nih.gov/pubmed/28058293
http://dx.doi.org/10.1021/acsomega.6b00356
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author Lee, Molly M.
Peterson, Blake R.
author_facet Lee, Molly M.
Peterson, Blake R.
author_sort Lee, Molly M.
collection PubMed
description [Image: see text] We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently found in proteins near ligand-binding sites, making this approach potentially applicable to a wide range of systems. To improve access to PB, we developed a scalable multigram synthesis of this fluorophore, starting with inexpensive 2,3,4,5-tetrafluorobenzoic acid. This route was used to synthesize fluorescent derivatives of biotin, as well as lower affinity thiobiotin, iminobiotin, and imidazolidinethione analogues that bind the protein streptavidin. Compared with previously published FRET acceptors for tryptophan, PB proved to be superior in both sensitivity and efficiency. These unique properties of PB enabled direct quantification of dissociation constants (K(d)) as well as competitive inhibition constants (K(i)) in the micromolar to nanomolar range. In comparison to analogous binding studies using fluorescence polarization, fluorescence quenching, or fluorescence enhancement, affinities determined using Trp-FRET were more precise and accurate as validated using independent isothermal titration calorimetry studies. FRET between tryptophan and PB represents a new tool for the characterization of protein–ligand complexes.
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spelling pubmed-52042062017-01-03 Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore Lee, Molly M. Peterson, Blake R. ACS Omega [Image: see text] We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently found in proteins near ligand-binding sites, making this approach potentially applicable to a wide range of systems. To improve access to PB, we developed a scalable multigram synthesis of this fluorophore, starting with inexpensive 2,3,4,5-tetrafluorobenzoic acid. This route was used to synthesize fluorescent derivatives of biotin, as well as lower affinity thiobiotin, iminobiotin, and imidazolidinethione analogues that bind the protein streptavidin. Compared with previously published FRET acceptors for tryptophan, PB proved to be superior in both sensitivity and efficiency. These unique properties of PB enabled direct quantification of dissociation constants (K(d)) as well as competitive inhibition constants (K(i)) in the micromolar to nanomolar range. In comparison to analogous binding studies using fluorescence polarization, fluorescence quenching, or fluorescence enhancement, affinities determined using Trp-FRET were more precise and accurate as validated using independent isothermal titration calorimetry studies. FRET between tryptophan and PB represents a new tool for the characterization of protein–ligand complexes. American Chemical Society 2016-12-19 /pmc/articles/PMC5204206/ /pubmed/28058293 http://dx.doi.org/10.1021/acsomega.6b00356 Text en Copyright © 2016 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Lee, Molly M.
Peterson, Blake R.
Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore
title Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore
title_full Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore
title_fullStr Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore
title_full_unstemmed Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore
title_short Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore
title_sort quantification of small molecule–protein interactions using fret between tryptophan and the pacific blue fluorophore
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204206/
https://www.ncbi.nlm.nih.gov/pubmed/28058293
http://dx.doi.org/10.1021/acsomega.6b00356
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