Mapping Protein-Protein Interfaces by MALDI Mass Spectrometry

最近进行的实验证明了使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）测量酰胺质子氢/氘交换率的可行性。该方法可以测量蛋白质表面非常快速交换的酰胺质子的交换速率。初步结果表明，如果酰胺在蛋白质-蛋白质相互作用的界面上，可能是由于溶剂排斥，快速交换的酰胺的交换速率降低。通过这种方式，可以快速绘制蛋白质-蛋白质界面。还有很多工作要做，以推广和量化该方法，以及了解溶剂可及性如何与蛋白质-蛋白质相互作用界面相关。实验计划要求首先确定影响蛋白质表面“覆盖”程度的变量和分析时间对氘保留的影响。其次，应确定复合物中离交换率与蛋白质浓度和结合亲和力相关的参数。第三，采用计算方法确定每个碎片上氘的数量，从而获得准确的动力学。第四，将开发快速混合淬灭方法来监测蛋白质络合时的构象变化。这个实验应该能得到络合过程中构象变化的“影像”。这种新方法对发现蛋白质复合物结构不可用的功能重要区域具有巨大的潜在影响。通过双杂交筛选可以快速识别新蛋白的相互作用界面。它也可以被证明是有用的筛选基因组信息到一个可行的规模。更重要的是，该方法提供了获取蛋白质-蛋白质界面动力学信息的途径。蛋白质-蛋白质界面对溶剂的接近程度如何？发生在界面上的溶剂可及性变化是否仅取决于界面的表面积，还是也取决于界面的疏水性？在蛋白质与蛋白质相互作用时，界面的某些区域是否变得不那么构象灵活？MALDI-TOF质谱方法有望在多种不同的蛋白质-蛋白质相互作用上回答这些问题，从而更好地理解控制蛋白质-蛋白质相互作用的生物物理参数。

Komives9808286Experiments have recently been performed that demonstate the feasibility of measuring hydrogen/deuterium exchange rates of amide protons using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The method permits measurement of the rate of exchange of the very rapidly exchanging amide protons on the surface of proteins. Preliminary results show that the exchange rates of the rapidly exchanging amides decrease if the amides are at the interface in a protein-protein interaction, presumably due to solvent exclusion. In this way, the protein-protein interface can be rapidly mapped. Much work remains to be done to generalize and quantify the method as well as to understand how solvent accessibility relates to protein-protein interaction interfaces. The experimental plan requires first that variables that affect the degree of "coverage" of the protein surface and the effect of analysis time on deuterium retention be determined. Second, the parameters that relate the rates of off-exchange in the complex to the protein concentrations and binding affinity shall be determined. Third, computational methods shall be implemented to ascertain the number of deuteriums on each fragment so that accurate kinetics can be obtained. Fourth, rapid mixing quench methods to monitor conformational changes upon protein-protein complexation will be developed. This experiment should yield a "movie" of the conformational changes occurring upon complexation.This novel method has tremendous potential impact for discovering the functionally important regions of proteins for which structures of their complexes are not available. The interacting interfaces of new proteins identified by the two-hybrid screen could be rapidly identified. It could also prove useful in culling genome information down to a workable size. More importantly, the method provides access to information about the dynamics of protein-protein interfaces. How accessible to the solvent is the protein-protein interface? Does the solvent accessibility change that occurs at the interface depend only on the surface area, or does it also depend on the hydrophobicity of the interface? Do certain regions of the interface become less conformationally flexible upon protein-protein interaction? The MALDI-TOF MS method shall be used to answer these questions on a variety of diverse protein-protein interactions so that the biophysical parameters that govern protein-protein interactions will be better understood.