Plenty of room to crystallize: swollen lipidic mesophases for improved and controlled in-meso protein crystallization

The lipidic cubic phase method for protein crystallization, also known as “in-meso”, has become widely recognized over the last decade, with clear advantages over other crystallization methods, especially when applied to membrane proteins. However, its advances are severely thwarted by the major limitation of the small size of the mesophase nanofluidic aqueous channels, preventing crystallization of large hydrophilic proteins and reconstitution of membrane proteins with large extramembrane domains. Here we show that modulation and increase of the size of the ordered mesophase hydrophilic domains, in a well-controlled way, by doping the hosting lipids with hydration-enhancing surfactants can open up the in-meso crystallization to a wide array of previously inaccessible proteins. To demonstrate unambiguously the potential of this strategy, we design as a hosting mesophase a reverse bicontinuous cubic phase of double diamond symmetry (Pn3m) with a well-defined and controllable channel diameter, by mixing monoglycerides and octyl glucoside surfactants, and show for the first time how this system can serve for the in-meso crystallization of the progressively larger β-lactoglobulin, thaumatin and elastase protein series.