Technical Highlight - March 2015
Short description: Using a membrane mimetic system, researchers produce properly folded integral membrane proteins that are suitable for subsequent X-ray crystallography analysis.
Denaturant-solubilized protein (left) is mixed with lipid to spontaneously assemble cubic mesophase (middle). Equilibration with denaturant-free buffer allows the protein to reconstitute into the bilayers as a consequence of a drop in denaturant concentration (right). 1
It is challenging to produce correctly folded membrane proteins for crystallography analyses and other experiments, as heterologous expression often yields aggregates or inclusion bodies. This problem can be solved by reconstituting the protein of interest in a membrane mimetic.
In this work, Li and Caffrey (PSI MPID) solubilize aggregates of diacylglycerol kinase (DgkA) by adding a denaturant and mixing it with lipid (monoacylglycerol). This results in spontaneous assembly of a lipid cubic phase, a bicontinuous, lyotropic liquid crystal or mesophase. This system consists of a curved bilayer with a multiple-branched, continuous aqueous channel on either side, an environment where proteins have been shown to be functional and fold with high efficiency. The addition of a denaturant-free buffer into this system causes the proteins to reconstitute into proximal lipid bilayers as the concentration of the denaturant drops.
DgkA renatured using this system was enzymatically active, as shown by thin layer chromatography analysis of the reaction product, lysophosphatidic acid. Following this confirmation, the researchers crystalized the protein in situ using the lipid cubic phase method and solved its X-ray structure with a resolution of 2.55 Å (PDB ID 4BRB).
The authors suggest that this system can also be used for vaccine development. This procedure is a rapid and inexpensive means of transforming protein precipitate into functional, bilayer-reconstituted integral membrane protein for numerous downstream applications.
D. Li & M. Caffrey Renaturing membrane proteins in the lipid cubic phase, a nanoporous membrane mimetic.
Sci Rep. 4, 5806 (2014). doi:10.1038/srep05806