Probing conformational changes by protein surface azidation

项目总结 尽管检测和表征构象的生物物理工具迅速出现 蛋白质结构的变化，研究蛋白质动力学在ITS中具有高灵敏度和可靠性 本土环境仍然是一个巨大的挑战。费力的样品制备和 对特殊设备的需求是使这些工具民主化的主要障碍。因此， 用于表征蛋白质构象动态变化的简单而健壮的平台高度 要求的。使用含叠氮的高价碘试剂，我们开发了一种新的 蛋白质表面叠氮质谱(ProSurA-MS) 它以无偏见的化学选择性检测蛋白质的构象变化。与 生物正交化学，ProSurA-MS允许蛋白质组范围的、特定位置的蛋白质图谱 覆盖范围广、重现性好的表面。ProSurA-MS有效地定位了构象 纯化蛋白质变性、蛋白质-小分子相互作用和蛋白质-蛋白质相互作用的变化 蛋白质相互作用。此外，ProSurA-MS检测到一种锌结合蛋白的结构变化 在锌耗竭时的全细胞裂解液中，并在活细胞中测量蛋白质组范围的氮化， 加强了复杂生物环境中蛋白质动力学的表征。这个 在此，建议的ProSurA-MS研究将使I)能够表征蛋白质的动态变化 翻译后修饰诱导的构象反应氧化应激和 监测金属转运蛋白不同遗传变异的蛋白质动力学(目标1)，二 对ProSurA反应和发展背后的化学机理的基本认识 具有更大叠氮产率和表面覆盖率的第二代试剂(目标2)和III) 一种鉴定蛋白质相互作用的新方法的建立 活细胞中的蛋白质表面氮化(目标3)。

PROJECT SUMMARY Despite the rapid emergence of biophysical tools to detect and characterize conformational changes in protein structure, studying protein dynamics with high sensitivity and reliability in its native environment remains a formidable challenge. Laborious sample preparation and requirement for special equipment present a major obstacle for democratizing these tools. Thus, a simple yet robust platform for characterizing dynamic changes in protein conformation is highly demanded. Using azide-containing hypervalent iodine reagents, we have developed a novel chemoproteomic platform termed Protein Surface Azidation Mass Spectrometry (ProSurA-MS) that detects conformational changes in proteins with unbiased chemoselectivity. Combined with bioorthogonal chemistry, ProSurA-MS allows proteome-wide, site-specific profiling of protein surfaces with wide coverage and reproducibility. ProSurA-MS effectively mapped conformational changes of purified proteins upon denaturation, protein-small molecule interaction, and protein- protein interaction. Additionally, ProSurA-MS detected structural changes in a zinc-binding protein in whole cell lysate upon zinc depletion and measured proteome-wide azidation in live cells, potentiating the characterization of protein dynamics in complex biological environments. The herein proposed ProSurA-MS studies will enable i) characterization of dynamic changes in protein conformation induced by post-translational modifications in response to oxidative stress and monitoring of the protein dynamics of different genetic variants of a metal transporter (Aim 1), ii) basic understanding of the chemical mechanism behind the ProSurA reaction and development of second generation reagents with greater azidation yield and surface coverage (Aim 2), and iii) establishment of a novel method for the identification of protein-protein interactions based on protein surface azidation in live cells (Aim 3).