Progress toward determining the Structure and Function of the Extra-cellular Domain of Glucagon Receptor and complexes with Ligands
College of Liberal Arts and Sciences
Physical Sciences and Mathematics
The G-protein coupled receptor (GPCR), or 7 Trans-membrane receptor (7TMR) family encompasses the largest number of membrane proteins in the human proteome. GPCRs are the target of about half of all pharmaceutical drugs on the market making them, and the determination of their atomic structure, one of the most important aspects of modern medicine. The current work applies the expression/purification system developed by our lab for other class B GPCRs whose structures have been reported to determine the structure of Glucagon receptor (GCGR). As in the past, the ECD is expressed as a fusion to maltose binding protein (MBP) in the oxidizing cytoplasm of an E. coli trxB gor host to allow disulfide bond formation. During purification, oxidized and reduced glutathione promote disulfide bond shuffling to maximize the yield of properly folded protein. Using this method GCGR was purified as an MBP fusion protein and Native gels confirmed a single species (folding scheme) had been purified for crystallizations. Although high resolution structures for the other mentioned GPCR ECDs have been determined, to date GCGR has still not been solved. Recent work on surface entropy reductions of the MBP have been found by other groups to facilitate crystallization of MBP fusion proteins. This approach led to an initial crystallization hit for GCGR and this constructs ability to bind ligand(s) was confirmed using an Alpha Screen assay. Progress on structural solution is presented.
National meeting of the American Crystallographic Association
Reynolds, Ross; Wren, L.; Parker, N.; Pioszak, A.; and Xu, E,, "Progress toward determining the Structure and Function of the Extra-cellular Domain of Glucagon Receptor and complexes with Ligands" (2013). Faculty Scholarly Dissemination Grants. 1086.