PROTEASE ACCESSIBILITY LADDERING: A PROTEOMIC TOOL FOR PROBING PROTEIN STRUCTURE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 有限蛋白水解法在生物化学和结晶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，由于获得足够数量的正确折叠的蛋白质和解释蛋白质降解的结果存在困难，该方法具有局限性。一种新的限制性蛋白水解法，称为蛋白酶可及性阶梯(PAL)，避免了这些并发症。在PAL中，标记的蛋白质在磁珠上以其自然折叠的状态进行纯化。当蛋白质附着在珠子上时，蛋白质会被蛋白酶探测到。用针对标签的抗体(例如，蛋白A、GFP和6xHis)洗脱蛋白水解性片段并通过免疫印迹进行检测。PAL可以很容易地检测蛋白质中的结构域边界和柔性环。PAL和比较蛋白结构建模的组合允许表征以前未知的结构(例如，Sec31，COPII包被小泡的一个成分)。PAL的高通量将极大地促进结构基因组和蛋白质组的研究。 我们目前正在开发用于MALDI-MS读出的PAL。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. We are currently developing PAL for use with MALDI-MS readout.