Crystallography of Membrane Proteins: From Crystallization to Structure
Although crystallographic studies of membrane proteins have progressed in the last 5 years, the field still remains challenging with several severe bottlenecks. The chapter focuses on the crystallization and describes two approaches, the classical vapor d
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1. Introduction Membrane proteins (MP) are the main functional units of membranes and represent roughly one-third of the proteins encoded in the genome. It has become clear in recent years that the study of membranes at the molecular level is of great importance not only in the deciphering of all cellular processes, but also in the understanding of the alterations leading to abnormal transformed cells and the action of the drugs. Indeed, 70% of drugs target membrane proteins. Although the number of membrane protein structures deposited since 1985, date of the first membrane protein structure (1), increases drastically it does not yet reach the rate achieved for soluble proteins (2). Currently, the RCSB Protein Data Bank contains more than 50,000 structures, among which less than 500 are Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_5, © Springer Science+Business Media, LLC 2010
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structures of membrane proteins (3). Major efforts undertaken in several laboratories in the last years as well as high-throughput crystallography offer a hope of correcting this imbalance. Nevertheless for the large-scale membrane protein structural biology to realize its full promise, significant challenges must be overcome. Two major bottlenecks await the experimentalist along the process toward the structure, the production of pure, stable and functional protein solubilized in amphiphiles, and the growth of well diffracting crystals. Most of the structural studies need to extract the proteins from the membrane using detergents. The matter is that the natural environment of these proteins is a lipid bilayer, and although detergents shield the hydrophobic surface of the protein from the solvent, they do not fully mimic membranes. Crystallization of a membrane protein should be undertaken only if sufficient biochemical and biophysical characterizations have been made. Solving the structure of a membrane protein by X-ray crystallography can encounter specific difficulties due to the detergent present in the crystals. This step is not defined as a major bottleneck but can prove to be difficult. This chapter describes the general strategy for solving membrane protein structures from the crystallization to the structure. In addition to the “classical” crystallization, it presents also alternate ways exploiting lipidic phases. Most of the crystallography is not specific to MPs, and only the main lines are indicated with references to other textbooks (4). The chapter focuses on aspects that are specific to MPs. All the steps described herein in a linear way, will not necessarily be followed sequentially. MPs very often crystallize with poor diffraction quality. In such cases, the procedure of getting good diffracting crystals and solving the structure will be an iterative process in which one has to cycle between the initial step of preparing the protein in a pure and soluble form for the crystallization,
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