Dokudovskaya Svetlana, Williams Rosemary, Devos Damien, Sali Andrej, Chait Brian T, Rout Michael P
Laboratory of Cellular and Structural Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Structure. 2006 Apr;14(4):653-60. doi: 10.1016/j.str.2006.02.006.
Limited proteolysis is widely used in biochemical and crystallographic studies to determine domain organization, folding properties, and ligand binding activities of proteins. The method has limitations, however, due to the difficulties in obtaining sufficient amounts of correctly folded proteins and in interpreting the results of the proteolysis. A new limited proteolysis method, named protease accessibility laddering (PAL), avoids these complications. In PAL, tagged proteins are purified on magnetic beads in their natively folded state. While attached to the beads, proteins are probed with proteases. Proteolytic fragments are eluted and detected by immunoblotting with antibodies against the tag (e.g., Protein A, GFP, and 6xHis). PAL readily detects domain boundaries and flexible loops within proteins. A combination of PAL and comparative protein structure modeling allows characterization of previously unknown structures (e.g., Sec31, a component of the COPII coated vesicle). PAL's high throughput should greatly facilitate structural genomic and proteomic studies.
有限蛋白水解在生物化学和晶体学研究中被广泛用于确定蛋白质的结构域组织、折叠特性和配体结合活性。然而，由于难以获得足够量的正确折叠的蛋白质以及难以解释蛋白水解的结果，该方法存在局限性。一种名为蛋白酶可及性阶梯分析（PAL）的新型有限蛋白水解方法避免了这些复杂问题。在PAL中，带标签的蛋白质在其天然折叠状态下通过磁珠进行纯化。附着在磁珠上时，用蛋白酶对蛋白质进行探测。蛋白水解片段被洗脱并用针对标签的抗体（如蛋白A、绿色荧光蛋白和6x组氨酸）进行免疫印迹检测。PAL能够轻松检测蛋白质中的结构域边界和柔性环。PAL与比较蛋白质结构建模相结合可对以前未知的结构（如COPII被膜小泡的一个组分Sec31）进行表征。PAL的高通量特性应能极大地促进结构基因组学和蛋白质组学研究。
