Time window expansion for HDX analysis of an intrinsically disordered protein.
Time window expansion for HDX analysis of an intrinsically disordered protein.
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DOI：
10.1007/s13361-013-0669-y
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发表时间：
2013-10
影响因子：
3.2
通讯作者：
Griffin, Patrick R.
中科院分区：
文献类型：
--
作者：
Goswami, Devrishi;Devarakonda, Srikripa;Chalmers, Michael J.;Pascal, Bruce D.;Spiegelman, Bruce M.;Griffin, Patrick R.
关键词：
由于未保护的酰胺氢与溶剂的快速交换，典型的HDX方法在检测固有无序蛋白质(IDP)方面的应用受到限制。IDP是一种天然无结构且在生理pH下高度动态的蛋白质。这些快速交换的酰胺的交换速率通常比典型的自动HDX-MS实验中使用的最短时间尺度(10s)更快。考虑到IDPs的功能重要性及其与许多疾病的关联，开发能够研究这些蛋白质的溶液动力学的方法以及探索IDPs与其广泛的结合伙伴的相互作用的能力是有价值的。在这里，我们报告了应用时间窗扩展到毫秒范围通过改变hdx实验的交换pH值来研究一个很好的特征的内源性dp；核受体共激活因子的激活区域，过氧化物体增殖物激活的受体γ共激活因子-1α(pgc-1α)。这种方法使定位pGC-1α的区域成为可能，这些区域是通过与核受体过氧化物酶体增殖物激活受体γ(PPARγ)的配体结合域(LBD)结合而稳定的。我们进一步证明了该方法对IDPGC-1α的其他结合伙伴的适用性，并为鉴定许多其他具有生物重要性的ID蛋白铺平了道路。
Application of typical HDX methods to examine intrinsically disordered proteins (IDP), proteins that are natively unstructured and highly dynamic at physiological pH, is limited due to the rapid exchange of unprotected amide hydrogens with solvent. The exchange rates of these fast exchanging amides are usually faster than the shortest time scale (10s) employed in typical automated HDX-MS experiments. Considering the functional importance of IDPs and their association with many diseases, it is valuable to develop methods that allow the study of solution dynamics of these proteins as well as the ability to probe the interaction of IDPs with their wide range of binding partners. Here, we report the application of time window expansion to the millisecond range by altering the on-exchange pH of the HDX experiment to study a well characterized IDP; the activation domain of the nuclear receptor coactivator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). This method enabled mapping the regions of PGC-1α that are stabilized upon binding the ligand binding domain (LBD) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). We further demonstrate the method’s applicability to other binding partners of the IDP PGC-1α and pave the way for characterizing many other biologically important ID proteins.
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影响因子：
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作者：
Burris TP;Busby SA;Griffin PR
通讯作者：
Griffin PR
DOI：
10.1016/j.jasms.2006.06.006
发表时间：
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影响因子：
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通讯作者：
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