ABSTRACT
Abstract ..................................................................................................................271 12.1 Introduction ................................................................................................272 12.2 Overexpression and Purification................................................................274 12.3 Detergent Selection....................................................................................276 12.4 Sample Preparation ....................................................................................281 12.5 Crystallization Methods.............................................................................282 12.6 Micelles and Bicelles.................................................................................283 12.7 Lipidic Mesophases ...................................................................................284 12.8 Bicelles, Lipopeptides, and Nanodiscs......................................................285 12.9 Crystallization ............................................................................................285 12.10 Case Study: Rhodopsin..............................................................................286 12.11 Conclusions ................................................................................................288 References..............................................................................................................288
Although the high-resolution crystal structure of bovine rhodopsin has been determined, many structure-function relationships remain to be learned from other GPCRs. Many pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergences from bovine rhodopsin and its related family members. Structure determination via x-ray crystallography of GPCRs can provide new avenues of engineering drugs with greater potencies and higher specificities. Since most membrane protein structures have been solved using x-ray crystallography, this method is most likely to be used for other GPCR structure elucidations. However, several obstacles must be overcome before this method becomes routine: overexpression of active GPCRs; solubilization and purification of active and stable GPCRs; and preparation of large, well-ordered crystals. This chapter outlines conditions for solubilization and purification using detergents and additives along with assessments of different crystallization formats of membrane proteins. It concludes
with a case study of the crystallization of rhodopsin and some of the challenges that remain with GPCR structures.
