A directed approach for engineering conditional protein stability using biologically silent small molecules
A directed approach for engineering conditional protein stability using biologically silent small molecules
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DOI：
10.1074/jbc.m703902200
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发表时间：
2007-08-24
影响因子：
4.8
通讯作者：
Wandless, Thomas J.
中科院分区：
文献类型：
--
作者：
Maynard-Smith, Lystranne A.;Chen, Ling-Chun;Wandless, Thomas J.
利用细胞渗透性分子调节特定蛋白质功能的能力可以成为询问生物系统的有力方法。为了使这种类型的“化学遗传”控制广泛的蛋白质，我们最近开发了一种实验系统，在该系统中，哺乳动物细胞中表达的小蛋白质结构域的稳定性取决于高亲和力配体的存在。这种依赖配体的稳定性被赋予任何融合的伙伴蛋白。fk506和雷帕霉素结合蛋白（FKBP12）已经成为广泛的生物物理分析的主题，包括对野生型蛋白以及数十个突变体的动力学和热力学研究。本研究的目的是确定在给定蛋白质内各种氨基酸取代的热力学稳定性（δ δ G(U-F)）是否可以预测工程中附加的配体依赖的不稳定结构域。我们使用FKBP12作为模型系统，发现体外热力学稳定性与突变体的细胞内降解率弱相关，并且给定突变使蛋白质不稳定的能力依赖于环境。我们评估了几种新的FKBP12配体稳定这些突变体的能力，发现一种称为Shield-1的细胞渗透性分子是最有效的稳定配体。然后，我们对不同浓度的Shield-1处理过的NIH3T3细胞进行了无偏置微阵列分析。这些研究表明，Shield-1不会引起明显的细胞反应。
The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506-and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (Delta Delta G(U-F)) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.