Membrane proteins play a vital role in many biological processes, and nearly 30% of proteins in eukaryotic cells are known to be membrane proteins. They have proven to be difficult to study due to their partially hydrophobic surfaces and lack of stability outside the lipid bilayer environment that is critical for membrane protein structure and activity. Crystallization of these proteins is notoriously challenging, which usually involves extensive detergent screening to identify optimal conditions.
Creative Biostructure takes advantage of our membrane screening platform for effective expression, solubilization, purification and crystallization of membrane proteins. For the past few years, our scientists have been actively developing and optimizing methods for the study of a variety of membrane proteins such as receptors, ion channels and transporters. The crystallization of membrane proteins generally begins with the test of a large number of possible crystallization reagents to identify the initial conditions, which are then subjected to further optimization to obtain well-diffracting crystals. Creative Biostructure has designed in-house 96-well sparse matrix screening systems specifically optimized for membrane proteins. We generally use the two major strategies to promote diffraction quality crystals:
Crystallization in surfo
This method involves the use surfactants to produce mixed micelles incorporating the target membrane protein, detergent and residual lipid if present. These water-soluble dispersions are treated in essentially the same way as soluble proteins for the production of crystals by vapor diffusion or microdialysis. The target protein can originate from native membranes or from the membranes or inclusion bodies of recombinant organisms. A variety of ionic, nonionic and zwitterionic detergents are screened during crystallization.
Crystallization in meso
Crystallization of membrane proteins can also be achieved in the presence of lipidic cubic phases or bicelles instead of detergent micelles. In both cases, an extended bilayer composed of lipid, detergent, and target protein is presumed to form to provide a native-like environment for membrane proteins.
Lipidic cubic phases (LCP) are mainly formed by mixing the selected lipid with an aqueous buffer at certain ratio and temperature, when the matrix becomes a transparent and non-birefringent gel-like material. In the three-dimensional continuous lipidic cubic phases such as the Pn3m phase, membrane proteins can freely diffuse in the lipid, instead of being enclosed in detergent micelle. The proteins molecules can therefore be concentrated to form well-ordered crystals.
Crystallization using bicelles is another successful in meso method. Bicelles are small bilayer disks formed from a number of lipid: amphiphile combinations when mixed at low temperature. They offer a native-like bilayer environment to the membrane proteins enhancing the growth of type I crystals. Crystallization in bicelles can be carried out in a traditional set up including the use of standard crystallization robotics and commercial crystallization screens.
Figure 1. Crystal packing arrangement and molecular structures of membrane proteins.
Our approaches aim at crystallizing membrane proteins while maintaining the native conformations and retaining their functions and activities. A great variety of biochemical, chemical and physical parameters are explored in the crystallization screening experiments, which have been fully automated at Creative Biostructure. Our efficient protocols and extensive process optimization greatly increase success rate, and we work closely with our customers to provide tailored strategies for the structure determination of membrane proteins of interest.
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