用于研究BCL-2家族中蛋白质相互作用的光交联方法。
Photocrosslinking Approach to Investigate Protein Interactions in the BCL-2 Family.
作者信息
Lin Jialing, Johnson Arthur E, Zhang Zhi
机构信息
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
出版信息
Methods Mol Biol. 2019;1877:131-149. doi: 10.1007/978-1-4939-8861-7_9.
The Bcl-2 family of proteins regulates mitochondrial outer membrane permeability thereby making life or death decisions for cells. Most of Bcl-2 proteins contain hydrophobic regions that are embedded in intracellular membranes such as mitochondria. These membrane proteins are difficult to express and purify thereby preluding biochemical and biophysical characterizations. Here, we describe a photocrosslinking approach based on in vitro synthesis of Bcl-2 proteins with photoreactive amino acid analogs incorporated at specific locations. These photoreactive proteins are reconstituted into liposomal membranes with defined phospholipids or mitochondrial membranes isolated from animals, and their interactions with other Bcl-2 proteins are detected by photocrosslinking.
Bcl-2蛋白家族调节线粒体外膜通透性，从而决定细胞的生死。大多数Bcl-2蛋白含有疏水区域，这些区域嵌入线粒体等细胞内膜中。这些膜蛋白难以表达和纯化，因此妨碍了生化和生物物理特性的表征。在这里，我们描述了一种光交联方法，该方法基于体外合成在特定位置掺入光反应性氨基酸类似物的Bcl-2蛋白。这些光反应性蛋白被重组到含有特定磷脂的脂质体膜或从动物分离的线粒体膜中，并且通过光交联检测它们与其他Bcl-2蛋白的相互作用。
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