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Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering
Lipidic cubic phase (LCP) crystallization methods have been essential in obtaining crystals of certain membrane proteins, particularly G-protein-coupled receptors. LCP crystallization is generally optimized across a large number of potential variables, one of which may be the choice of the solubiliz...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
International Union of Crystallography
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792994/ https://www.ncbi.nlm.nih.gov/pubmed/33520240 http://dx.doi.org/10.1107/S2052252520013974 |
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author | Cleveland IV, Thomas Blick, Emily Krueger, Susan Leung, Anna Darwish, Tamim Butler, Paul |
author_facet | Cleveland IV, Thomas Blick, Emily Krueger, Susan Leung, Anna Darwish, Tamim Butler, Paul |
author_sort | Cleveland IV, Thomas |
collection | PubMed |
description | Lipidic cubic phase (LCP) crystallization methods have been essential in obtaining crystals of certain membrane proteins, particularly G-protein-coupled receptors. LCP crystallization is generally optimized across a large number of potential variables, one of which may be the choice of the solubilizing detergent. A better fundamental understanding of the behavior of detergents in the LCP may guide and simplify the detergent selection process. This work investigates the distribution of protein and detergent in LCP using the membrane protein bacteriorhodopsin (bR), with the LCP prepared from highly deuterated monoolein to allow contrast-matched small-angle neutron scattering. Contrast-matching allows the scattering from the LCP bilayer itself to be suppressed, so that the distribution and behavior of the protein and detergent can be directly studied. The results showed that, for several common detergents, the detergent micelle dissociates and incorporates into the LCP bilayer essentially as free detergent monomers. In addition, the detergent octyl glucoside dissociates from bR, and neither the protein nor detergent forms clusters in the LCP. The lack of detergent assemblies in the LCP implies that, upon incorporation, micelle sizes and protein/detergent interactions become less important than they would be in solution crystallization. Crystallization screening confirmed this idea, with crystals obtained from bR in the presence of most detergents tested. Thus, in LCP crystallization, detergents can be selected primarily on the basis of protein stabilization in solution, with crystallization suitability a lesser consideration. |
format | Online Article Text |
id | pubmed-7792994 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
spelling | pubmed-77929942021-01-29 Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering Cleveland IV, Thomas Blick, Emily Krueger, Susan Leung, Anna Darwish, Tamim Butler, Paul IUCrJ Research Papers Lipidic cubic phase (LCP) crystallization methods have been essential in obtaining crystals of certain membrane proteins, particularly G-protein-coupled receptors. LCP crystallization is generally optimized across a large number of potential variables, one of which may be the choice of the solubilizing detergent. A better fundamental understanding of the behavior of detergents in the LCP may guide and simplify the detergent selection process. This work investigates the distribution of protein and detergent in LCP using the membrane protein bacteriorhodopsin (bR), with the LCP prepared from highly deuterated monoolein to allow contrast-matched small-angle neutron scattering. Contrast-matching allows the scattering from the LCP bilayer itself to be suppressed, so that the distribution and behavior of the protein and detergent can be directly studied. The results showed that, for several common detergents, the detergent micelle dissociates and incorporates into the LCP bilayer essentially as free detergent monomers. In addition, the detergent octyl glucoside dissociates from bR, and neither the protein nor detergent forms clusters in the LCP. The lack of detergent assemblies in the LCP implies that, upon incorporation, micelle sizes and protein/detergent interactions become less important than they would be in solution crystallization. Crystallization screening confirmed this idea, with crystals obtained from bR in the presence of most detergents tested. Thus, in LCP crystallization, detergents can be selected primarily on the basis of protein stabilization in solution, with crystallization suitability a lesser consideration. International Union of Crystallography 2021-01-01 /pmc/articles/PMC7792994/ /pubmed/33520240 http://dx.doi.org/10.1107/S2052252520013974 Text en © Thomas Cleveland IV et al. 2021 http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Research Papers Cleveland IV, Thomas Blick, Emily Krueger, Susan Leung, Anna Darwish, Tamim Butler, Paul Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering |
title | Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering |
title_full | Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering |
title_fullStr | Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering |
title_full_unstemmed | Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering |
title_short | Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering |
title_sort | direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering |
topic | Research Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792994/ https://www.ncbi.nlm.nih.gov/pubmed/33520240 http://dx.doi.org/10.1107/S2052252520013974 |
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