Mapping Protein-Protein Interfaces by MALDI Mass Spectrometry

通过 MALDI 质谱绘制蛋白质-蛋白质界面

• 批准号: 9808286
• 负责人: Elizabeth Komives
• 金额: $ 27万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808286&HistoricalAwards=false
• 关键词: Mapping; Protein; Interfaces; MALDI; Mass

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.

最近进行的Komives9808286实验表明，用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)测量酰胺质子的氢/氚交换率是可行的。该方法允许测量蛋白质表面非常快速地交换的酰胺质子的交换率。初步结果表明，在蛋白质-蛋白质相互作用中，如果酰胺处于界面处，则快速交换的酰胺的交换率降低，这可能是由于溶剂排斥。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有关。实验计划要求首先确定影响蛋白质表面“覆盖”程度的变量，以及分析时间对氢保留的影响。其次，应确定将复合体中的非交换率与蛋白质浓度和结合亲和力相关的参数。第三，应该采用计算方法来确定每个碎片上的氘的数量，以便能够获得准确的动力学。第四，将开发快速混合猝灭方法来监测蛋白质-蛋白质络合的构象变化。这项实验应该会产生一部关于络合时发生的构象变化的电影。这种新的方法对于发现蛋白质的功能重要区域具有巨大的潜在影响，而这些区域的络合物结构是不可用的。通过双杂交筛选鉴定的新蛋白的相互作用界面可以被快速识别。它在筛选基因组信息到可行的大小方面也可能被证明是有用的。更重要的是，该方法提供了有关蛋白质-蛋白质界面动力学的信息。蛋白质-蛋白质界面对溶剂的可及性有多大？发生在界面上的溶剂可及性变化是仅取决于表面积，还是还取决于界面的疏水性？当蛋白质-蛋白质相互作用时，界面的某些区域会不会变得不那么灵活？MALDI-TOF MS方法应用于回答各种不同蛋白质-蛋白质相互作用的问题，以便更好地了解支配蛋白质-蛋白质相互作用的生物物理参数。