Direct Localization of Detergents and Bacteriorhodopsin in the Lipidic Cubic Phase by Small-Angle Neutron Scattering

Published

October 19, 2020

Author(s)

Thomas Cleveland, Emily Blick, Susan T. Krueger, Anna Leung, Tamim A. Darwish, Paul Butler

Abstract

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.

Citation

IUCrJ

Pub Type

Journals

Download Paper

DOI Link

Keywords

lipidic cubic phase, lcp, small-angle neutron scattering, sans, lipid, membrane, protein

Citation

Cleveland, T. , Blick, E. , Krueger, S. , Leung, A. , Darwish, T. and Butler, P. (2020), Direct Localization of Detergents and Bacteriorhodopsin in the Lipidic Cubic Phase by Small-Angle Neutron Scattering, IUCrJ, [online], https://doi.org/10.1107/S2052252520013974 (Accessed November 23, 2024)