| G protein-coupled receptors comprise the largest family of cell surface receptors, and are responsible for the internalization of extracellular stimuli. They play important roles in many physiological processes, and as such are the target of about 30% of currently marketed drugs. As integral membrane proteins, the structural study of GPCRs by X-ray crystallography has been limited due to the difficulties associated with crystallization. However, in recent years the development of different protein engineering methods has resulted in the acceleration of crystallizing many different GPCR targets. One such method has been the replacement of the third intracellular loop of the receptor with T4 lysozyme, thereby increasing the surface area for crystal lattice contacts. Despite the success of this method, it has thus far been limited to only the use of T4 lysozyme, and the utility of other fusion protein partners for this purpose has not been explored. To expand the repertoire of engineering methods that can be used to crystallize GPCRs, the Protein Data Bank was searched for other proteins with promising characteristics for making GPCR fusions. From a panel of candidates, we selected apocytochrome b562RIL and were able to crystallize three GPCRs by using it as an alternative fusion protein partner: the A2A adenosine receptor, the beta-2-adrenergic receptor, and the nociceptin receptor. This thesis presents the screening methodology and new structural insights obtained from the resulting GPCR structures. In particular, A2AAR is the highest resolution GPCR structure to date (1.8 angstrom resolution), and NOP is a new structure of the opioid receptor family of GPCRs, both of which provide many new biological insights to this class of important receptors. |
