PROTEASE ACCESSIBILITY LADDERING: A PROTEOMIC TOOL FOR PROBING PROTEIN STRUCTURE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.