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).

项目总结