Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering.

Thomas Cleveland Iv,Tamim Darwish,Anna Leung,Paul Butler,Susan Krueger,Emily Blick

doi:10.1107/s2052252520013974

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.

Highlights

Incorporation of membrane proteins into lipidic mesophases such as the lipidic cubic phase (LCP) has become an important method for obtaining crystals for structural studies
A convincing example of this observation can be found in the work by Cherezov et al (2006), where crystals of the lightharvesting II complex grown in surfo require space in the lattice for the detergent belt around the transmembrane region; whereas in meso crystals of the same protein allow for close packing between transmembrane regions of adjacent proteins
We initially measured small-angle neutron scattering (SANS) from D2O micellar solutions for a panel of 32 detergents (Fig. S1 and Table S1 of the supporting information) taken from common classes used in membrane protein biochemical and crystallization studies

Summary

Introduction

Incorporation of membrane proteins into lipidic mesophases such as the lipidic cubic phase (LCP) has become an important method for obtaining crystals for structural studies. This procedure is often referred to as ‘in meso’ crystallization, in contrast with ‘in surfo’, which refers to direct solution crystallization of the protein/detergent complex. When membrane proteins are crystallized from detergent solution, a belt of detergent similar to a micelle remains bound around the hydrophobic region of the membrane protein, and must be accommodated by the crystal lattice. Upon incorporation into the LCP, the micelle is generally assumed to dissociate (Caffrey, 2000), allowing crystal contacts and packing to be mediated by transmembrane regions of the protein as well. A recent review that discusses these ideas is available (Birch et al, 2018)

Methods

Results

Conclusion