Correlation between ligand-receptor affinity and the transcription readout in a yeast three-hybrid system
Correlation between ligand-receptor affinity and the transcription readout in a yeast three-hybrid system
复制标题
DOI：
10.1021/bi049716n
复制
发表时间：
2004-08-17
期刊：
影响因子：
2.9
通讯作者：
Cornish, VW
中科院分区：
文献类型：
--
作者：
de Felipe, KS;Carter, BT;Cornish, VW
酵母双杂交技术已被证明是一种检测蛋白质间相互作用和构建全基因组蛋白质相互作用图谱的有效方法。最近，三杂交测定已经出现作为检测蛋白质-RNA和蛋白质-小分子相互作用的手段。尽管常规使用的双杂交测定和三杂交系统的潜力，一直很少有定量表征，以了解如何与转录激活的蛋白质相互作用的强度。目前尚不清楚三混合动力系统中的额外相互作用是否会损害系统的灵敏度。因此，在这里，我们着手确定小分子三杂交系统的K-D截止值，并确定K-D与转录激活水平之间是否存在相关性。设计了FK 506结合蛋白12（FKBP 12）的一系列突变，以改变该蛋白对FK 506结合蛋白12（SLF）小分子合成配体的亲和力。这些FKBP 12变体被过表达和纯化，并且使用荧光偏振测定法测量它们对SLF的K-D。然后使用lacZ报告基因测定这些变体在Mtx-DHFR酵母三杂交系统中的转录激活水平。Mtx酵母三杂交系统的K-D截断值约为0.000000。50 nM。此外，转录激活水平与结合相互作用的强度相关，尽管动态范围仅为1个数量级。这些结果表明，三杂交试验具有必要的灵敏度药物发现。然而，小的动态范围突出了基于亲和力区分相互作用的基于平衡的测定的局限性。
The yeast two-hybrid assay has proven to be a powerful method to detect protein-protein interactions as well as to derive genome-wide protein interaction maps. More recently, three-hybrid assays have emerged as a means to detect both protein-RNA and protein-small molecule interactions. Despite the routine use of the two-hybrid assay and the potential of three-hybrid systems, there has been little quantitative characterization to understand how the strength of the protein interaction correlates with transcription activation. It is not known if the additional interaction in three-hybrid systems compromises the sensitivity of the system. Thus, here, we set out to determine the K-D cutoff of a small molecule three-hybrid system and to determine if there is a correlation between the K-D and the levels of transcription activation. A series of mutations to FK506-binding protein 12 (FKBP12) were designed to vary the affinity of this protein for the small molecule synthetic ligand for FK506-binding protein 12 (SLF). These FKBP12 variants were overexpressed and purified, and their K-D's for SLF were measured using a fluorescence polarization assay. Then the levels of transcription activation in a Mtx-DHFR yeast three-hybrid system were determined for these variants using a lacZ reporter gene. The K-D cutoff of the Mtx yeast three-hybrid system is found to be ca. 50 nM. Further, the levels of transcription activation correlate with the strength of the binding interaction, though the dynamic range is only 1 order of magnitude. These results establish that the three-hybrid assay has the requisite sensitivity for drug discovery. However, the small dynamic range highlights a limitation to equilibrium-based assays for discriminating interactions based on affinity.