Ignatova Zoya, Gierasch Lila M
Max Planck Institute for Biochemistry, Martinsried, Germany.
Methods Enzymol. 2007;428:355-72. doi: 10.1016/S0076-6879(07)28021-8.
Nature has developed many strategies to ensure that the complex and challenging protein folding reaction occurs in vivo with adequate efficiency and fidelity for the success of the organism. Among the strategies widely employed in a huge range of species and cell types is the elaboration of small organic molecules called osmolytes that offset the potentially damaging effects of osmotic stress. While considerable knowledge has been gained in vitro regarding the influence of osmolytes on protein structure and folding, it is of great interest to probe the effects of osmolytes in cells. We have developed an in-cell fluorescent-labeling method that enables the study of protein stability and also protein aggregation in vivo. We utilize a genetically encoded tag called a tetra-Cys motif that binds specifically to a bis-arsenical fluorescein-based dye "FlAsH"; we inserted the tetra-Cys motif into a protein of interest in such a way that the FlAsH signal reported on the state of folding or aggregation of the protein. Then, we designed protocols to assess how various osmolytes influence the stability and propensity to aggregate of our protein of interest. These are described here. Not only are there potential biotechnological applications of osmolytes in the quest to produce greater quantities of well-folded proteins, but also osmolytes may serve as tools and points of departure for therapeutic intervention in protein folding and aggregation diseases. Having in vivo methods to analyze how osmolytes affect folding and aggregation enhances our ability to further these goals greatly.
自然界已经发展出许多策略，以确保复杂且具有挑战性的蛋白质折叠反应在体内以足够的效率和保真度发生，从而使生物体得以成功生存。在众多物种和细胞类型中广泛采用的策略之一是合成称为渗透溶质的小分子有机化合物，这些化合物可以抵消渗透应激的潜在破坏作用。虽然在体外已经获得了大量关于渗透溶质对蛋白质结构和折叠影响的知识，但探究渗透溶质在细胞中的作用仍具有重大意义。我们开发了一种细胞内荧光标记方法，该方法能够在体内研究蛋白质稳定性以及蛋白质聚集情况。我们利用一种名为四半胱氨酸基序的基因编码标签，它能特异性结合一种基于双砷荧光素的染料 “FlAsH”；我们将四半胱氨酸基序插入到感兴趣的蛋白质中，使得 FlAsH 信号能够反映该蛋白质的折叠或聚集状态。然后，我们设计了实验方案来评估各种渗透溶质如何影响我们感兴趣的蛋白质的稳定性和聚集倾向。这些内容将在此处进行描述。渗透溶质不仅在生产更多折叠良好的蛋白质方面具有潜在的生物技术应用，而且还可能作为蛋白质折叠和聚集疾病治疗干预的工具和出发点。拥有体内分析渗透溶质如何影响折叠和聚集的方法，极大地增强了我们实现这些目标的能力。
