During the last few years, there has been an extraordinary increase in publications describing the manifold applications of monoolein, one of the most important lipids in the fields of drug delivery, emulsion stabilization and protein crystallization. In this perspective we present a comprehensive review of the phase behavior of this ‘magic lipid’. An account of various mesophases formed in the presence of water and a collection of formulae for the calculation of their nano-structural parameters are provided. Effects of chemical and biological molecules including lipids, detergents, salts, sugars, proteins and DNA on the classical behavior are also discussed. Physicochemical triggers such as, temperature, pressure and shearing modulate the phase behavior of monoolein self assemblies that are covered in subsequent sections. Finally the growing applications of monoolein in various fields are also reported.

Monoolein: a magic lipid?

Mimicking the Cell: Bio-Inspired Functions of Supramolecular Assemblies

The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published structures that have been obtained using in meso-grown crystals are given. Recommendations for conducting the screening process to give a more productive outcome are summarized. The fact that the in meso method also works with soluble proteins should not be overlooked. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The review ends with a view to the future and to the bright prospects for the method, which continues to contribute to our understanding of the molecular mechanisms of some of nature's most valued proteinaceous robots.

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A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes

Cubosomes: remarkable drug delivery potential

The principal route to determine the structure and the function and interactions of membrane proteins is via macromolecular crystallography. For macromolecular crystallography to be successful, structure-quality crystals of the target protein must be forthcoming, and crystallogenesis represents a major challenge. Several techniques are employed to crystallize membrane proteins, and the bulk of these techniques make direct use of solubilized protein-surfactant complexes by the more traditional, so-called in surfo methods. An alternative in meso approach, which employs a bicontinuous lipidic mesophase, has emerged as a method with considerable promise in part because it involves reconstitution of the solubilized protein back into a stabilizing and organizing lipid bilayer reservoir as a prelude to crystallogenesis. A hypothesis for how the method works at the molecular level and experimental evidence in support of the proposal are reviewed here. The latest advances, successes, and challenges associated with the method are described.

Crystallizing Membrane Proteins for Structure Determination: Use of Lipidic Mesophases

A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization

The aqueous phase behavior of phytantriol (PT) in mixtures of monoolein (MO), distearoylphosphatidylglycerol (DSPG), propylene glycol (PG), polyethylene glycol 400 (PEG 400) and 2-methyl-2,4-pentanediol (MPD) was investigated by visual inspection, polarized light microscopy and small angle X-ray diffraction at room temperature. The phase diagrams of PT and MO in water are qualitatively very similar and PT/MO mixtures in excess water form one cubic phase of space group Pn3m irrespective of mixing ratio. The addition of the charged membrane lipid DSPG to the PT system gives rise to a considerable water swelling of the cubic phases as well as the occurrence of a cubic phase of space group Im3m. Whereas all three solvents studied give rise to a sponge (L3) phase in the MO-water system, this phase was only found when MPD was added to the PT-water system. The results are discussed with respect to the chemical differences between PT and MO.

Aqueous self-assembly of phytantriol in ternary systems: effect of monoolein, distearoylphosphatidylglycerol and three water-miscible solvents.

The phase behavior of 1-glyceryl monooleyl ether (GME) in mixtures of propylene glycol (PG) and water was investigated by visual inspection, polarization microscopy, small-angle X-ray diffraction, and conductance measurements. A phase diagram, based on over 200 samples of the ternary system GME-PG-water, was constructed at 20 degrees C. Without PG, GME forms a reverse micellar phase with up to 10 wt % water and a reverse hexagonal liquid-crystalline phase between 10 and 25 wt % water, a phase that can coexist with excess water. If PG is added in amounts exceeding about 10 wt %, then cubic and lamellar liquid-crystalline phases start to form. A cubic phase, belonging to space group Pn3m, can coexist with excess PG-water mixtures. If even more PG is added, then the cubic phase is transformed into a sponge phase. A lamellar phase forms at water contents between 10 and 15 wt % and with widely differing PG/GME weight ratios. We postulate that the phase behavior is caused by the fact that PG makes the interfacial region between self-assembled GME and PG-water less negatively curved, which in turn allows for the formation of the new phases. The phase behavior obtained for the GME system shows a striking similarity with the phase behavior of the corresponding system in which the GME has been replaced by the ester, 1-glycerol monooleate (GMO), differing only in one extra carbonyl oxygen. The major difference is the lower amount of water present in the GME phases, an effect that is mainly due to the more hydrophobic character of GME compared to that of GMO.

Pia Wadsten-Hindrichsen

Papers

A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization

Aqueous self-assembly of phytantriol in ternary systems: effect of monoolein, distearoylphosphatidylglycerol and three water-miscible solvents.

Cubic, sponge, and lamellar phases in the glyceryl monooleyl ether-propylene glycol-water system.