Senisterra Guillermo A, Ghanei Hamed, Khutoreskaya Galina, Dobrovetsky Elena, Edwards Aled M, Privé Gilbert G, Vedadi Masoud
Structural Genomics Consortium, Toronto, Ontario, Canada.
J Biomol Screen. 2010 Mar;15(3):314-20. doi: 10.1177/1087057109357117. Epub 2010 Feb 11.
Protein stabilization upon ligand binding has frequently been used to identify ligands for soluble proteins. Methods such as differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) have been employed in the 384-well format and have been useful in identifying ligands that promote crystallization and 3D structure determination of proteins. However, finding a generic method that is applicable to membrane proteins has been a challenge as the high hydrophobicity of membrane proteins and the presence of detergents essential for their solubilization interfere with fluorescence-based detections. Here the authors used MsbA (an adenosine triphosphate binding cassette transporter), CorA (a Mg(++) channel), and CpxA (a histidine kinase) as model proteins and show that DSLS is not sensitive to the presence of detergents or protein hydrophobicity and can be used to monitor thermodenaturation of membrane proteins, assess their stability, and detect ligand binding in a 384-well format.
配体结合后蛋白质的稳定化作用常被用于识别可溶性蛋白质的配体。诸如差示扫描荧光法（DSF）和差示静态光散射法（DSLS）等方法已被应用于384孔板形式，并且在识别促进蛋白质结晶和三维结构测定的配体方面很有用。然而，找到一种适用于膜蛋白的通用方法一直是一项挑战，因为膜蛋白的高疏水性以及溶解它们所必需的去污剂的存在会干扰基于荧光的检测。在这里，作者使用MsbA（一种三磷酸腺苷结合盒转运蛋白）、CorA（一种Mg(++)通道）和CpxA（一种组氨酸激酶）作为模型蛋白，并表明DSLS对去污剂的存在或蛋白质疏水性不敏感，可用于监测膜蛋白的热变性、评估其稳定性以及在384孔板形式中检测配体结合。
