New methods are provided for the post-genomic era that will permit the analysis of the dynamic expression and localization of gene products in living cells. Herein we propose the development of such a method from a bioorganic approach involving organic synthesis and protein engineering. Specifically, novel compounds bearing two maleimide groups attached directly to fluorescent cores will be prepared, whose latent fluorescence is quenched until their maleimide groups undergo a specific thiol addition reaction. Complementary a-helical proteins are designed bearing two cysteine residues appropriately positioned to react with our novel fluorogens. Genetically fusing our helical probe peptides to test proteins of interest, we can selectively label the target sequence in living cells with our small synthetic fluorogenic molecules. The scope of this technique is described in the context of studying protein localization and protein-protein interactions in living cells.
提供了新的方法用于后
基因组时代,这些方法将允许分析
基因产物在活细胞中的动态表达和定位。在这里,我们提出了从
生物有机
化学方法出发的一种方法的发展,涉及有机合成和蛋白工程。具体来说,将制备带有两个马来
酰亚胺基团直接连接到荧光核心的新化合物,其潜在荧光在其马来
酰亚胺基团经历特定巯基加成反应之前被熄灭。设计了具有两个半胱
氨酸残基的互补α-螺旋蛋白,这些残基被适当地定位以与我们的新型荧光基团发生反应。通过遗传融合我们的螺旋探针肽到感兴趣的蛋白质上,我们可以使用我们的小型合成荧光基团在活细胞中选择性地标记目标序列。在研究蛋白质在活细胞中的定位和蛋白质相互作用方面描述了这种技术的范围。