Abstract
The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.
Highlights
Lipidic cubic phases and related amphiphile-rich materials have served as matrices for growing a variety of membrane protein crystals [1], the latter of which were used in determining X-ray crystallographic structures of several high-impact target proteins such as G-protein coupled receptors [2,3,4,5]
We investigate the early stages of this new crystallization regime, namely the incorporation of the membrane protein RC (Photosynthetic Reaction Center from Rhodobacter sphaeroides) into a lipidic phase prior to crystallization
2 Optimized RC/monoolein pre-incubation time In order to devise a simplified PLI membrane protein crystallization protocol [17], we investigated the effect of the duration of the RC solution exposure to the lipid phase (Figure 4)
Summary
Lipidic cubic phases and related amphiphile-rich materials have served as matrices for growing a variety of membrane protein crystals [1], the latter of which were used in determining X-ray crystallographic structures of several high-impact target proteins such as G-protein coupled receptors [2,3,4,5]. A procedure employing positive displacement devices for the preparation of crystallization experiments in dedicated crystallization plates was introduced [10], later reproduced [11,12], and refined with the goal to further reduce setup volumes and increase expediency [13]. Most of these technological developments aimed at improving the tools that manipulate small volumes of the highly viscous LCP (lipidic cubic phase) that is obtained when monoolein is mixed with a membrane protein solution [14]. Attempts have been made to avoid the requirement for dealing with the highly viscous
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