Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

中文翻译：

---

氢-氘交换和羟基自由基足迹，用于绘制人类溴结构域与小分子抑制剂的疏水相互作用。

基于质谱 (MS) 的蛋白质足迹是绘制蛋白质-配体相互作用的一种有价值的结构工具，已广泛应用于蛋白质-蛋白质复合物，在绘制大型界面方面取得了成功。在这里，我们利用了一种结合氢-氘交换（HDX）和羟基自由基足迹（即蛋白质的快速光化学氧化（FPOP））的综合足迹策略，对含有人类溴结构域的蛋白质 4（BRD4）的相互作用进行了分子水平表征。 ) 与疏水性苯二氮卓抑制剂。HDX 没有为结合界面的位置提供强有力的证据，可能是因为小分子对溶剂的屏蔽作用不大。相反，HDX 表明 BRD4 似乎通过显示由结合引起的动力学适度下降而稳定。相比之下，FPOP 指向疏水腔中的关键结合区域，也通过晶体学鉴定，因此在绘制 BRD4 与化合物 1 的相互作用时表现出比 HDX 更高的灵敏度。 在没有或在低浓度自由基清除剂的情况下，FPOP 修饰Met 残基显示出显着差异，反映了蛋白质构象的微小变化。这个问题可以通过使用足够量的适当清除剂来避免，正如由羟基自由基剂量计指导的 FPOP 动力学所暗示的那样。Met 残基上的 FPOP 修饰显示出显着差异，反映了蛋白质构象的微小变化。这个问题可以通过使用足够量的适当清除剂来避免，正如由羟基自由基剂量计指导的 FPOP 动力学所暗示的那样。Met 残基上的 FPOP 修饰显示出显着差异，反映了蛋白质构象的微小变化。这个问题可以通过使用足够量的适当清除剂来避免，正如由羟基自由基剂量计指导的 FPOP 动力学所暗示的那样。