Effects of 2‑monoacylglycerol on in meso crystallization and the crystal structures of integral membrane proteins

Monoacylglycerols (MAGs) are the lipids most commonly used for crystallizing membrane proteins by the in meso method. The acyl chain and glycerol components of MAGs are ester-linked at the sn-1 hydroxyl of glycerol in 1-MAGs. 1-MAGs succumb to spontaneous transacylation, where the chain migrates between the sn-1 and sn-2 hydroxyls. In the mesophase formed by 1-monoolein, the most commonly used MAG, equilibrium is reached in 2 weeks at 6 mol % of 2-monoolein. Variability in 2-MAG content appears in synthetic MAGs produced for the purpose of host lipid screening. In the interests of reproducibility, extensive purification has been implemented to minimize the 2-MAG content of the newly synthesized lipid. Here we show, with two membrane proteins, that such efforts are not needed. Specifically, the initial 2-MAG content can vary over wide limits without affecting negatively the crystallization process or the structures obtained with those crystals. Dispensing with rounds of purification means that MAGs can be produced using protocols that are simpler, faster, less expensive, and more environmentally friendly. An added feature of having 2-monoolein in the crystallization mix is that it stabilizes mesophases with larger aqueous channels, well suited to the structure determination of proteins and complexes with large extramembrane domains.