Fooksman David R, Edidin Michael, Barisas B George
Department of Chemistry, Colorado State University, Ft. Collins, CO 80523, USA.
Biophys Chem. 2007 Oct;130(1-2):10-6. doi: 10.1016/j.bpc.2007.06.013. Epub 2007 Jul 6.
Clustering of membrane proteins is a dynamic process which can regulate cellular function and signaling. The size of receptor and other membrane protein clusters can in principle be measured in terms of their rotational diffusion. However, in practice, measuring rotation of membrane proteins of live cells has been difficult, largely because of the difficulty of rigidly attaching reporter groups to the molecules of interest. Here we show that polarized photobleaching recovery can detect rotation of membrane proteins genetically tagged with yellow fluorescent protein, YFP. MHC class I molecules were engineered with a rigid, in-sequence, YFP tag followed at the C-terminus by a pair of crosslinkable domains. When crosslinker was added we could detect changes in rotational anisotropy decay consistent with clustering of the MHC molecules. This result points the way to use of engineered fluorescent fusion proteins to measure rotational diffusion in native cell membranes.
膜蛋白的聚集是一个动态过程，可调节细胞功能和信号传导。原则上，受体和其他膜蛋白簇的大小可以通过其旋转扩散来测量。然而，在实际操作中，测量活细胞中膜蛋白的旋转一直很困难，主要是因为难以将报告基团牢固地连接到感兴趣的分子上。在这里，我们表明偏振光漂白恢复可以检测用黄色荧光蛋白（YFP）进行基因标记的膜蛋白的旋转。MHC I类分子经过工程改造，带有一个刚性的、按顺序排列的YFP标签，在C端接着一对可交联结构域。加入交联剂后，我们可以检测到与MHC分子聚集一致的旋转各向异性衰减的变化。这一结果为利用工程荧光融合蛋白测量天然细胞膜中的旋转扩散指明了方向。
