Quantifying Protection in Disordered Proteins Using Millisecond Hydrogen Exchange-Mass Spectrometry and Peptic Reference Peptides
Quantifying Protection in Disordered Proteins Using Millisecond Hydrogen Exchange-Mass Spectrometry and Peptic Reference Peptides
复制标题
DOI：
10.1021/acs.biochem.6b01312
复制
发表时间：
2017-08-08
期刊：
影响因子：
2.9
通讯作者：
Weis, David D.
中科院分区：
文献类型：
--
作者：
Al-Naqshabandi, Mohammed A.;Weis, David D.
内在无序蛋白质（IDP）中瞬时结构的程度和位置为其构象集合提供了有价值的见解，并可以更好地理解耦合结合和折叠。毫秒酰胺氢交换（HX）可以提供这样的信息，但它是难以量化的瞬态结构化的程度。一个原因是，瞬时无序蛋白质经历FIX的速率仅略低于酰胺HX通过非结构化无规卷曲的速率，即化学HX速率。在这项工作中，我们评估了几种不同的方法来获得适用于无序蛋白质的毫秒氢交换质谱（HX-MS）分析的化学FIX速率的准确模型：（1）使用Englander [Bai，Y.，等人（1993）Proteins 17，75-86]，（2）在6 M尿素或3 M氯化胍存在下测量FIX，和（3）通过直接来源于目的蛋白的肽片段测量FIX。首先，使用非结构化的模型肽和无序结构域的甲状腺和类维生素A受体的激活剂和CREB结合蛋白作为模型IDPs，我们表明，英格兰人的方法有轻微的不准确性，导致低估的化学交换率。第二，模型肽的HX-MS测量显示FIX速率被高浓度的变性剂显著改变。第三，我们发现，HX的参考肽从感兴趣的蛋白质的测量提供了最准确的方法来量化的程度，在无序的蛋白质的瞬时结构毫秒EX-MS。
The extent and location of transient structure in intrinsically disordered proteins (IDPs) provide valuable insights into their conformational ensembles and can lead to a better understanding of coupled binding and folding. Millisecond amide hydrogen exchange (HX) can provide such information, but it is difficult to quantify the degree of transient structuring. One reason is that transiently disordered proteins undergo FIX at rates only slightly slower than the rate of amide HX by an unstructured random coil, the chemical HX rate. In this work, we evaluate several different methods of obtaining an accurate model for the chemical FIX rate suitable for millisecond hydrogen exchange mass spectrometry (HX-MS) analysis of disordered proteins: (1) calculations using the method of Englander [Bai, Y., et al. (1993) Proteins 17, 75-86], (2) measurement of FIX in the presence of 6 M urea or 3 M guanidinium chloride, and (3) measurement of FIX by peptide fragments derived directly from the proteins of interest. First, using unstructured model peptides and disordered domains of the activator for thyroid and retinoid receptors and the CREB binding protein as the model IDPs, we show that the Englander method has slight inaccuracies that lead to underestimation of the chemical exchange rate. Second, HX-MS measurements of model peptides show that FIX rates are changed dramatically by high concentrations of the denaturant. Third, we find that measurements of HX by reference peptides from the proteins of interest provide the most accurate approach for quantifying the extent of transient structure in disordered proteins by millisecond EX-MS.