Tsirigotaki A, Papanastasiou M, Trelle M B, Jørgensen T J D, Economou A
KU Leuven, Rega Institute for Medical Research, Laboratory of Molecular Bacteriology, Leuven, Belgium.
IMBB-FoRTH, Iraklio, Greece.
Methods Enzymol. 2017;586:57-83. doi: 10.1016/bs.mie.2016.09.028. Epub 2016 Nov 8.
Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.
蛋白质折叠是活细胞中一个复杂而精确的过程。大多数分泌蛋白避免在细胞质中折叠，被靶向运输到膜上，然后转运进入/穿过膜。它们的靶向和转运能力状态是未正确折叠的。然而，一旦它们到达合适的细胞区室，就可以折叠成天然状态。由于无序度增加、蛋白质大小、聚集倾向以及观察时间尺度等因素常常限制X射线晶体学和核磁共振等典型结构分析方法的应用，前体蛋白的未正确折叠状态在结构上仍缺乏充分表征。在此，我们提出一种用于体外分析未折叠的具有转运能力的蛋白质状态并将其与天然状态进行比较的替代方法。我们利用氢/氘交换结合质谱法（HDX-MS），这是一种基于结构化与非结构化蛋白质状态/区域以及高动态区域与更刚性区域中不同同位素交换速率的方法。我们展示了一个完整的结构表征流程，从多肽的制备到数据分析与解读。讨论了用于分析标记蛋白的蛋白酶解和质谱条件，随后是HDX-MS数据的分析与解读。我们强调了HDX-MS在识别高度柔性蛋白质状态中短结构化区域方面的适用性，我们实验室通过一个分泌蛋白实例进行了实验验证。最后，我们讨论了比较HDX-MS中的统计分析。该方案适用于任何蛋白质和蛋白质大小，无论其转运能力的丧失是缓慢还是快速。它可以很容易地适用于更复杂的组装体，例如伴侣蛋白与未正确折叠蛋白质状态的相互作用。
