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Synthetic single domain antibodies for the conformational trapping of membrane proteins

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate...

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Autores principales: Zimmermann, Iwan, Egloff, Pascal, Hutter, Cedric AJ, Arnold, Fabian M, Stohler, Peter, Bocquet, Nicolas, Hug, Melanie N, Huber, Sylwia, Siegrist, Martin, Hetemann, Lisa, Gera, Jennifer, Gmür, Samira, Spies, Peter, Gygax, Daniel, Geertsma, Eric R, Dawson, Roger JP, Seeger, Markus A
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967865/
https://www.ncbi.nlm.nih.gov/pubmed/29792401
http://dx.doi.org/10.7554/eLife.34317
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author Zimmermann, Iwan
Egloff, Pascal
Hutter, Cedric AJ
Arnold, Fabian M
Stohler, Peter
Bocquet, Nicolas
Hug, Melanie N
Huber, Sylwia
Siegrist, Martin
Hetemann, Lisa
Gera, Jennifer
Gmür, Samira
Spies, Peter
Gygax, Daniel
Geertsma, Eric R
Dawson, Roger JP
Seeger, Markus A
author_facet Zimmermann, Iwan
Egloff, Pascal
Hutter, Cedric AJ
Arnold, Fabian M
Stohler, Peter
Bocquet, Nicolas
Hug, Melanie N
Huber, Sylwia
Siegrist, Martin
Hetemann, Lisa
Gera, Jennifer
Gmür, Samira
Spies, Peter
Gygax, Daniel
Geertsma, Eric R
Dawson, Roger JP
Seeger, Markus A
author_sort Zimmermann, Iwan
collection PubMed
description Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.
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spelling pubmed-59678652018-05-29 Synthetic single domain antibodies for the conformational trapping of membrane proteins Zimmermann, Iwan Egloff, Pascal Hutter, Cedric AJ Arnold, Fabian M Stohler, Peter Bocquet, Nicolas Hug, Melanie N Huber, Sylwia Siegrist, Martin Hetemann, Lisa Gera, Jennifer Gmür, Samira Spies, Peter Gygax, Daniel Geertsma, Eric R Dawson, Roger JP Seeger, Markus A eLife Biochemistry and Chemical Biology Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins. eLife Sciences Publications, Ltd 2018-05-24 /pmc/articles/PMC5967865/ /pubmed/29792401 http://dx.doi.org/10.7554/eLife.34317 Text en © 2018, Zimmermann et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Zimmermann, Iwan
Egloff, Pascal
Hutter, Cedric AJ
Arnold, Fabian M
Stohler, Peter
Bocquet, Nicolas
Hug, Melanie N
Huber, Sylwia
Siegrist, Martin
Hetemann, Lisa
Gera, Jennifer
Gmür, Samira
Spies, Peter
Gygax, Daniel
Geertsma, Eric R
Dawson, Roger JP
Seeger, Markus A
Synthetic single domain antibodies for the conformational trapping of membrane proteins
title Synthetic single domain antibodies for the conformational trapping of membrane proteins
title_full Synthetic single domain antibodies for the conformational trapping of membrane proteins
title_fullStr Synthetic single domain antibodies for the conformational trapping of membrane proteins
title_full_unstemmed Synthetic single domain antibodies for the conformational trapping of membrane proteins
title_short Synthetic single domain antibodies for the conformational trapping of membrane proteins
title_sort synthetic single domain antibodies for the conformational trapping of membrane proteins
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967865/
https://www.ncbi.nlm.nih.gov/pubmed/29792401
http://dx.doi.org/10.7554/eLife.34317
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