Integral membrane proteins are arguably the most important class of proteins in terms of their role in cellular interactions and their associated significance in disease pathways. Although they account for one-fourth of the human genome, less than 300 independent structures for membrane proteins have been solved.1 In comparison, there are more than 60,000 solved structures of soluble proteins in the Protein Data Bank.1 The relatively slow pace of structure determination for membrane proteins is primarily due to the difficulties encountered in forming crystals. The amphiphilic nature of membrane proteins introduces challenges associated with isolating and stabilizing these proteins in the absence of their in vivo membrane environments.
In the face of these obstacles, Lipidic Cubic Phase (LCP) has emerged as an effective medium for crystallizing membrane proteins. LCP is a liquid crystal that spontaneously forms upon the mixing of lipids with water and generates an inviting environment for membrane proteins. The bicontinuous bilayer formed by LCP mimics the cellular membrane and thus stabilizes the proteins, enabling crystallization. The general method of crystallizing proteins utilizing LCP was first introduced by Landau & Rosenbusch in 1996, and has resulted in the determination of the structures of 30 unique membrane proteins.2,3 (see http://cherezov.scripps.edu/structures.htm for a count of all proteins crystallized in LCP)
The current pace of adoption of LCP-based methods has increased substantially, in spite of the steep learning curve associated with their implementation. Recent advances in automation technology for drop setting, condition screening, and imaging have made LCP crystallization considerably more accessible.
This blog series will discuss the three major steps of LCP crystallization and how automation technology is significantly facilitating the use of this powerful methodology.
1. Raman, P.; Cherezov, V.; Caffrey C., The membrane protein data bank. Cellular and Molecular Life Sciences 2006, 63, 34-51.
2. Landau, E.M., and J.P. Rosenbusch. Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci.1996 93: 14532-14535.
3. Caffrey M and Cherezov V (2009) Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4: 706-731.