Optimization of Constructs for Expression, Purification and Crystallization of Glutamate Receptor Ion Channels

Abstract

Eukaryotic membrane protein purification is a daunting task limited by protein expression at quantities sufficient for structural analysis. Membrane proteins constitute 30% of the genome encoded by eukaryotes, however, to date, only 300 independent structures of integral membrane proteins have been reported, and many of these are from prokaryotes. Glutamate receptor ion channels (iGluRs) with their large extracellular and disordered cytoplasmic C-terminal domains pose an even greater challenge for crystallographic studies. To date only one full-length iGluR structure has been solved, for the homomeric GluR2 AMPA receptor subtype at 3.6-A resolution1. Although this provides the first insights into glutamate receptor structure and subunit arrangement, it is a single snapshot of an antagonist bound conformation of the closed state of the ion channel. To further understand the mechanisms of receptor function, structures in different conformational states will be needed. Further, full-length structures for other iGluR subtypes such as kainate and NMDA have yet not been structurally explored till date. Towards this goal, using fluorescence-detection size-exclusion chromatography (FSEC)2, as a rapid pre-crystallization screening method we have optimized the GluR6 (GluK2) and GluR7 (GluK3) kainate receptor constructs for crystallographic studies. We have systematically optimized constructs for both subtypes, which required deletion of residues at the C-terminus, mutation of free cysteines, and knock out of N-linked glycosylation sites to improve its stability and crystallization behavior. The constructs were screened for expression level, monodispersity and stability of the ion channel tetramer. Screening of optimal buffer conditions, detergents and lipids has yielded very promising leads for crystallization, and has established that the proteins can be maintained as stable tetramers in detergent solutions for periods exceeding a week.1.Sobolevsky AI, ROSconi MP, Gouaux E. (2009). Nature 462:745-756.2.Kawate, T, Gouaux, E. (2006). Structure 14:673-681.