Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Cleveland IV, Thomas, Blick, Emily, Krueger, Susan, Leung, Anna, Darwish, Tamim, Butler, Paul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2021
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
_version_ 1783633897878192128
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
work_keys_str_mv AT clevelandivthomas directlocalizationofdetergentsandbacteriorhodopsininthelipidiccubicphasebysmallangleneutronscattering
AT blickemily directlocalizationofdetergentsandbacteriorhodopsininthelipidiccubicphasebysmallangleneutronscattering
AT kruegersusan directlocalizationofdetergentsandbacteriorhodopsininthelipidiccubicphasebysmallangleneutronscattering
AT leunganna directlocalizationofdetergentsandbacteriorhodopsininthelipidiccubicphasebysmallangleneutronscattering
AT darwishtamim directlocalizationofdetergentsandbacteriorhodopsininthelipidiccubicphasebysmallangleneutronscattering
AT butlerpaul directlocalizationofdetergentsandbacteriorhodopsininthelipidiccubicphasebysmallangleneutronscattering