title: A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening.
abstract: Optimization of membrane protein stability under different solution conditions is essential for obtaining crystals that diffract to high resolution. Traditional methods that evaluate protein stability require large amounts of material and are, therefore, ill suited for medium- to high-throughput screening of membrane proteins. Here we present a rapid and efficient fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS). In this method, the target protein is fused to GFP. Heated protein samples, treated with a panel of additives, are then analyzed by FSEC. FSEC-TS allows one to evaluate the thermostability of nanogram-to-microgram amounts of the target protein under a variety of conditions without purification. We applied this method to the Danio rerio P2X4 receptor and Caenorhabditis elegans GluCl to screen ligands, ions, and lipids, including newly designed cholesterol derivatives. In the case of GluCl, the screening results were used to obtain crystals of the receptor in the presence of lipids.
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, OR 97239, USA.
authors.forename: Motoyuki
authors.initials: M
authors.lastname: Hattori
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Hibbs
authors.forename: Eric
authors.initials: E
authors.lastname: Gouaux
mesh_terms: D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018118:Chloride Channels / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000302:isolation & purification; D008055:Lipids / Q000737:chemistry; D011485:Protein Binding; D055550:Protein Stability; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D044366:Transition Temperature; D029961:Zebrafish Proteins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; D008055:Lipids; D058848:Receptors, Purinergic P2X4; D011993:Recombinant Fusion Proteins; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; C109777:glutamate-gated chloride channels; D049452:Green Fluorescent Proteins