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首页> 外文期刊>Biochemistry >Use of amide exchange mass spectrometry to study conformational changes within the endopolygalacturonase II-homogalacturonan-polygalacturonase inhibiting protein system.
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Use of amide exchange mass spectrometry to study conformational changes within the endopolygalacturonase II-homogalacturonan-polygalacturonase inhibiting protein system.

机译：使用酰胺交换质谱研究内聚半乳糖醛酸酶II-同型半乳糖醛酸聚糖-聚半乳糖醛酸酶抑制蛋白系统内的构象变化。

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摘要

Amide exchange mass spectrometry (MS) was used to study the enzyme endopolygalacturonase II (EPG-II) from Aspergillus niger as it binds to an oligosaccharide substrate. A localized decrease in the level of deuterium incorporation in EPG-II of the EPG-II-oligosaccharide complex relative to that of the free EPG-II identified the location of substrate contact, which is in agreement with published site specific mutation studies. In addition, when bound with substrate, regions of EPG-II remote from the substrate binding site became exposed to the solvent, as revealed by an increase in the amount of incorporated deuterium, indicating a conformational change in the enzyme. Fluorescence experiments were performed to provide additional evidence for an altered conformation of EPG-II as a result of substrate binding. This novel application of amide exchange-MS to the study of protein-carbohydrate binding has, for the first time, described in detail the conformational changes associated with EPG-II when it binds a substrate. Amide exchange-MS was also used to study the interactions of EPG-II and the polygalacturonase inhibitor protein (PGIP). Mass spectral data of the EPG-II-oligosaccharide complex in the presence of Phaseolus vulgaris PGIP indicate that the inhibitor contacts EPG-II at a site remote from the substrate binding cleft, and is restricting the conformational changes of EPG-II. Fluorescence experiments also revealed that upon binding of PGIP, the conformational changes mentioned above for the EPG-II-substrate complex are minimized. These results, together with previously reported data, point to a location on EPG-II for interaction with PGIP as well as a possible mechanism for noncompetitive inhibition of EPG-II.
机译：使用酰胺交换质谱（MS）研究来自黑曲霉的内聚半乳糖醛酸内切酶II（EPG-II），因为它与寡糖底物结合。相对于游离的EPG-II，EPG-II-寡糖复合物的EPG-II中氘掺入水平的局部降低确定了底物接触的位置，这与已发表的位点特异性突变研究一致。另外，当与底物结合时，远离底物结合位点的EPG-II区域变得暴露于溶剂，这由掺入的氘量的增加所揭示，表明酶的构象变化。进行荧光实验以提供另外的证据，作为底物结合的结果，EPG-II构象的改变。酰胺交换-MS在研究蛋白质与碳水化合物结合方面的这种新颖应用首次详细描述了与EPG-II结合底物时的构象变化。酰胺交换-MS还用于研究EPG-II和聚半乳糖醛酸酶抑制剂蛋白（PGIP）的相互作用。在菜豆PGIP存在下EPG-II-寡糖复合物的质谱数据表明，抑制剂在远离底物结合裂隙的位点接触EPG-II，并限制了EPG-II的构象变化。荧光实验还表明，结合PGIP后，上述针对EPG-II-底物复合物的构象变化被最小化。这些结果与先前报道的数据一起指向了EPG-II上与PGIP相互作用的位置，以及非竞争性抑制EPG-II的可能机制。

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