Ashok Yashwanth, Jaakola Veli-Pekka
Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Oulu 90014, Finland.
MethodsX. 2016 Mar 14;3:212-8. doi: 10.1016/j.mex.2016.03.009. eCollection 2016.
Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.
膜蛋白在去污剂中通常不稳定。因此,在脂质环境中对膜蛋白进行生化和生物物理研究可提供适合膜蛋白的近乎天然的环境。然而,对嵌入脂质双层中的蛋白质进行操作仍然很困难。诸如纳米圆盘和脂质立方相等方法已被开发出来,以便于对膜蛋白进行操作,并对膜蛋白有了重要的认识。传统上,纳米圆盘中受体的功能重建是用放射性配体进行研究的。我们提出了一种简单且更快的方法,用于在使用放射性配体初步优化合适条件后,对蛋白质批次进行常规表征,以研究重组膜蛋白的功能。该方法的优点是:•评估膜蛋白功能重建的更快且通用的方法。•适用于高通量形式(≥96孔板形式)。•在近乎天然的脂质环境中进行稳定性测量以及脂质依赖性解链温度测量。