Membrane Protein Structure and Dynamics Methods and Protocols
Membrane proteins play key roles in numerous cellular processes, in particular mediating cell-to-cell communication and signaling events that lead to a multitude of biological effects. Membrane proteins have also been implicated in many critical diseases
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1. Introduction 1.1. Crystallization of Membrane Proteins
Despite many heroic efforts and some success stories, the number of membrane protein structures determined continues to lag far behind soluble proteins. One major hurdle is the difficulty in obtaining good-quality crystals. For many membrane proteins, detergents are the partner that they “cannot live with, or without.” Detergents are necessary for the extraction and solubilization of membrane proteins, while at the same time they can interfere with crystal growth. By their nature, detergents cover much of the hydrophobic surface of a membrane protein leaving little surface area for crystal growth. In the absence of a sizable soluble domain, the protein contacts required for crystal growth are limited to mostly loop regions. This often leads to poor crystal quality since loops are typically the more flexible portion of the protein (1). This challenging task of growing well-ordered membrane protein crystals has prompted the development of a number of technical
Nagarajan Vaidehi and Judith Klein-Seetharaman (eds.), Membrane Protein Structure and Dynamics: Methods and Protocols, Methods in Molecular Biology, vol. 914, DOI 10.1007/978-1-62703-023-6_1, © Springer Science+Business Media, LLC 2012
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S. Agah and S. Faham
advances. These developments include the antibody method (2), the protein fusion method (3, 4), and the lipidic cubic phase (LCP) method (5). These methods have proven useful, even critical in certain cases. Nonetheless, a universal solution appears out of reach as the unique features of each protein can considerably influence the crystallization process. The distinctive features of each protein may favor a certain method over others; thus the development and advancement of various approaches are beneficial. The success of the LCP method demonstrated that membrane proteins can be crystallized from lipid media (5), not just from detergent media. The lipid cubic phase forms a network of interconnected bilayers that is generated by the lipid monoolein (1-cis-9-octadecenoyl)rac-glycerol. Due to inherent challenges of the LCP method, such as its high viscosity, we explored the utility of a different lipid phase, namely, bicelles, for the crystallization of membrane proteins, and developed the bicelle method described here. 1.2. Bicelles
Bicelles are bilayer micelles that are formed by specific mixtures of a lipid and a detergent (Fig. 1). Bicelles were originally used in solid-state NMR studies and continue to receive much attention in the NMR field, since they tend to partially align in a magnetic field (6). We were successful in crystallizing bacteriorhodopsin in bicelles (7), and determined its structure from two different bicelle-forming
Fig. 1. Representation of the morphology of bicelles. Bicelles are bilayer discs. The bilayer is formed by the lipid (DMPC), with the detergent (CHAPSO) covering the edges of the hydrophobic bilayer.
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Crystallization of Membrane Proteins in Bicelles
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compositions, demonstrating that bicelles can be a
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