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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...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2018
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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. |
format | Online Article Text |
id | pubmed-5967865 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
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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