Design of ultra-swollen lipidic mesophases for the crystallization of membrane proteins with large extracellular domains

Abstract

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3–5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d→Pn3m→Ia3d and Pn3m→Im3m→Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology.

Nature‐Inspired Design and Application of Lipidic Lyotropic Liquid Crystals

“…The described structures contain domains in the size range of a few nm, which are hydrophilic, lipophilic, and amphiphilic. It has been reported that the addition of charged phospholipids can swell inverse bicontinuous cubic phases from lattice parameters close to 10 nm up to values in the region of 50 nm …”

“…Although in meso crystallization has revolutionized the field of membrane structural biology by providing a robust strategy for the crystallization of membrane proteins, the method has for a long time remained restricted to membrane proteins with small (<5 nm) extracellular and intracellular domains, so that the structure of most membrane proteins with large extra‐ and intracellular domains remains unsolved to date. A recent work by Zabara et al however has significantly extended the reach of in meso protein crystallization, by using ultraswollen lipidic mesophases, designed to allow reconstitution and crystallization of membrane proteins with large extra and intracellular domains. The authors used a combination of neutral and charged lipids to design ultraswollen bicontinuous cubic phases of double gyroid (), double diamond (), and double primitive () structures, five times larger than traditional lipidic mesophases.…”

Bicontinuous cubic phases in biological and artificial self-assembled systems

“…Therefore, scaling artificial fabrication is a huge challenge encountered by scientists. It has been reported that inverse bicontinuous cubic phases can be formed when charged phospholipids are introduced, with the unit cell parameter increased from 10 to 50 nm [164][165][166]. Therefore, the formation of inverse micelles could be a promising method for creating materials containing a lattice size comparable to biological cubic membranes, especially those associated with structural colors.…”

SCryPTA:A web-based platform for analyzing Small-Angle Scattering curves of lyotropic liquid crystals

“…In particular, LLC structures can be mainly classified into different phases, as micellar, lamellar, cubic, and hexagonal according to their structures 4,5 . LLC are found in vivo 6,7 , they can be used in the crystallization of membrane proteins 8,9 , control of chemical reaction in food 10 and as drug delivery systems [11][12][13][14] .…”

“…In vivo, LLC structures are found in alveolar surface of mammalian lung 6 , products of digested oil droplets in small intestine 7 , thylakoid membrane in plant chloroplast 7 , intracellular membranes in various cell organelles 8 , wing scales or cuticle of green butterflies or beetles 9 . Some examples are applications in materials science, medical and food fields as: crystallization of proteins [10][11][12] , DNA, SiRNA encapsulation 13 , burst release for medical application 14 , increase of bioavailability of lipophilic or amphiphilic compounds and control of chemical reaction (oxidation and Maillard chain reaction) in food 15,16 .…”