Probing conformational changes by protein surface azidation

通过蛋白质表面叠氮化探测构象变化

• 批准号: 10630213
• 负责人: Alexander Adibekian
• 金额: $ 33.58万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630213
• 关键词: Acetylation; Amino Acids; Azides; Binding; Biological; Biophysics; Buffers; Cells; Chemicals; Chemistry; Co-Immunoprecipitations; Complex; Coupled; Cysteine; Development; Disease; Ensure; Environment; Enzymes; Equipment; Generations; Housekeeping; Investigation; Iodine; Ion Transport; Maps; Mass Spectrum Analysis; Measures; Mediating; Membrane Proteins; Metals; Methods; Modeling; Molecular Conformation; Monitor; Oxidative Regulation; Oxidative Stress; Peptides; Phosphines; Post-Translational Protein Processing; Preparation; Protein Conformation; Protein Dynamics; Proteins; Proteome; Reaction; Reagent; Reporting; Reproducibility; Sampling; Site; Solubility; Special Equipment; Surface; System; Techniques; Technology; Testing; Zinc; aqueous; biophysical tools; chemoproteomics; crosslink; design; detection method; disulfide bond; endopeptidase Clp; genetic variant; improved; interest; novel; protein profiling; protein protein interaction; protein purification; protein structure; response; small molecule; synthetic peptide; tool; zinc-binding protein

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).

项目摘要 尽管生物物理工具迅速出现，以检测和表征构象 蛋白质结构的变化，研究蛋白质动力学具有高灵敏度和可靠性， 自然环境仍然是一个巨大的挑战。费力的样品制备和 对专用设备的要求是这些工具民主化的主要障碍。因此，在本发明中， 一个简单而强大的平台，用于表征蛋白质构象的动态变化， 他问道。使用含叠氮化物的高价碘试剂，我们开发了一种新的 蛋白质表面叠氮化质谱（ProSurA-MS） 以无偏的化学选择性检测蛋白质的构象变化。结合 生物正交化学，ProSurA-MS允许蛋白质组范围内的，位点特异性的蛋白质分析 具有广泛的覆盖面和再现性的表面。ProSurA-MS有效地映射了构象 纯化的蛋白质在变性、蛋白质-小分子相互作用和蛋白质- 蛋白质相互作用此外，ProSurA-MS检测到锌结合蛋白的结构变化 在锌耗尽后的全细胞裂解物中和在活细胞中测量的蛋白质组范围的叠氮化中， 加强复杂生物环境中蛋白质动力学的表征。的 本文提出的ProSurA-MS研究将使i）表征蛋白质的动态变化 通过响应氧化应激的翻译后修饰诱导的构象， 监测金属转运蛋白的不同遗传变体的蛋白质动力学（Aim 1），ii） 基本了解ProSurA反应和开发背后的化学机制 具有更大叠氮化产率和表面覆盖率的第二代试剂（目标2），以及iii） 建立一种新的蛋白质-蛋白质相互作用的鉴定方法， 活细胞中的蛋白质表面叠氮化（Aim 3）。