PROTEASE ACCESSIBILITY LADDERING: A PROTEOMIC TOOL FOR PROBING PROTEIN STRUCTURE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 有限蛋白水解广泛用于生物化学和晶体学研究，以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和解释蛋白水解的结果，该方法具有局限性。一种新的有限的蛋白水解方法，称为蛋白酶可及性阶梯（PAL），避免了这些并发症。在PAL中，标记的蛋白质在其天然折叠状态下在磁珠上纯化。当附着在珠子上时，蛋白质被蛋白酶探测。蛋白水解片段被洗脱并通过用针对标签的抗体（例如，蛋白A、GFP和6xHis）。PAL容易检测蛋白质内的结构域边界和柔性环。PAL和比较蛋白质结构建模的组合允许表征先前未知的结构（例如，Sec 31，COPII包被囊泡的组分）。PAL的高通量将极大地促进结构基因组学和蛋白质组学研究。

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.