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Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids

[Image: see text] We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine...

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Autores principales: Naganathan, Saranga, Ray-Saha, Sarmistha, Park, Minyoung, Tian, He, Sakmar, Thomas P., Huber, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310623/
https://www.ncbi.nlm.nih.gov/pubmed/25524496
http://dx.doi.org/10.1021/bi501267x
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author Naganathan, Saranga
Ray-Saha, Sarmistha
Park, Minyoung
Tian, He
Sakmar, Thomas P.
Huber, Thomas
author_facet Naganathan, Saranga
Ray-Saha, Sarmistha
Park, Minyoung
Tian, He
Sakmar, Thomas P.
Huber, Thomas
author_sort Naganathan, Saranga
collection PubMed
description [Image: see text] We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine receptor 5 (CCR5) at site-specific amber codon mutations. We then used strain-promoted azide–alkyne [3+2] cycloaddition to label the azF-CCR5 variants with a FLAG peptide epitope-conjugated aza-dibenzocyclooctyne (DBCO) reagent. A microtiter plate-based sandwich fluorophore-linked immunosorbent assay was used to probe simultaneously the FLAG epitope and the receptor using infrared dye-conjugated antibodies so that the extent of DBCO incorporation, corresponding nominally to labeling efficiency, could be quantified ratiometrically. The extent of incorporation of DBCO at the various sites was evaluated in the context of a recent crystal structure of maraviroc-bound CCR5. We observed that labeling efficiency varied dramatically depending on the topological location of the azF in CCR5. Interestingly, position 109 in transmembrane helix 3, located in a hydrophobic cavity on the extracellular side of the receptor, was labeled most efficiently. Because the bioorthogonal labeling and detection strategy described might be used to introduce a variety of different peptide epitopes or fluorophores into engineered expressed receptors, it might prove to be useful for a wide range of applications, including single-molecule detection studies of receptor trafficking and signaling mechanism.
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spelling pubmed-43106232015-12-19 Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids Naganathan, Saranga Ray-Saha, Sarmistha Park, Minyoung Tian, He Sakmar, Thomas P. Huber, Thomas Biochemistry [Image: see text] We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine receptor 5 (CCR5) at site-specific amber codon mutations. We then used strain-promoted azide–alkyne [3+2] cycloaddition to label the azF-CCR5 variants with a FLAG peptide epitope-conjugated aza-dibenzocyclooctyne (DBCO) reagent. A microtiter plate-based sandwich fluorophore-linked immunosorbent assay was used to probe simultaneously the FLAG epitope and the receptor using infrared dye-conjugated antibodies so that the extent of DBCO incorporation, corresponding nominally to labeling efficiency, could be quantified ratiometrically. The extent of incorporation of DBCO at the various sites was evaluated in the context of a recent crystal structure of maraviroc-bound CCR5. We observed that labeling efficiency varied dramatically depending on the topological location of the azF in CCR5. Interestingly, position 109 in transmembrane helix 3, located in a hydrophobic cavity on the extracellular side of the receptor, was labeled most efficiently. Because the bioorthogonal labeling and detection strategy described might be used to introduce a variety of different peptide epitopes or fluorophores into engineered expressed receptors, it might prove to be useful for a wide range of applications, including single-molecule detection studies of receptor trafficking and signaling mechanism. American Chemical Society 2014-12-19 2015-01-27 /pmc/articles/PMC4310623/ /pubmed/25524496 http://dx.doi.org/10.1021/bi501267x Text en Copyright © 2014 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 Naganathan, Saranga
Ray-Saha, Sarmistha
Park, Minyoung
Tian, He
Sakmar, Thomas P.
Huber, Thomas
Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids
title Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids
title_full Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids
title_fullStr Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids
title_full_unstemmed Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids
title_short Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids
title_sort multiplex detection of functional g protein-coupled receptors harboring site-specifically modified unnatural amino acids
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310623/
https://www.ncbi.nlm.nih.gov/pubmed/25524496
http://dx.doi.org/10.1021/bi501267x
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