TY - JOUR
T1 - A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening
AU - Hattori, Motoyuki
AU - Hibbs, Ryan E.
AU - Gouaux, Eric
N1 - Funding Information:
We thank A. Penmatsa for providing the lipid screening kit for GluCl, J. Michel for technical help, and L. Vaskalis for assistance with figures. This work was supported by the Uehara Memorial Foundation (M.H.), the American Asth Foundation (E.G.), and the National Institutes of Health (R.H. and E.G.). E.G. is an investigator with the Howard Hughes Medical Institute.
PY - 2012/8/8
Y1 - 2012/8/8
N2 - 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.
AB - 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.
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U2 - 10.1016/j.str.2012.06.009
DO - 10.1016/j.str.2012.06.009
M3 - Article
C2 - 22884106
AN - SCOPUS:84864853832
SN - 0969-2126
VL - 20
SP - 1293
EP - 1299
JO - Structure with Folding & design
JF - Structure with Folding & design
IS - 8
ER -