Spatially resolved DNP- assisted NMR illuminates the conformational ensemble of α- synuclein in intact viable cells

• 成果类型: Article
• 署名作者: Kragelj, Jaka; Ghosh, Rupam; Xiao, Yiling; Dumarieh, Rania; Lagasca, Dominique; Krishna, Sakshi; Frederick, Kendra K.
• 署名单位: University of Texas System; University of Texas Southwestern Medical Center; University of Texas System; University of Texas Southwestern Medical Center; National Institute of Chemistry - Slovenia
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13567
• DOI: 10.1073/pnas.2500367122
• 发表日期: 2025-06-10
• 关键词: dynamic nuclear-polarization; n-terminal acetylation; secondary structure; proteins; spectroscopy; binding; membranes; efficient; affinity; reveals

摘要

The protein alpha- syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an alpha- helical- rich membrane- associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of alpha- syn for membranes, evidence that the alpha- helical form is adopted inside cells has been indirect. DNP- assisted solid- state NMR on frozen cellular samples can report directly on the entire conformational ensemble. By controlling the distribution of the DNP agent throughout the cellular biomass, such experiments can provide quantitative information upon the entire structural ensemble or provide information about spatially resolved subpopulations. When the polarization agent is dispersed homogeneously throughout the cell, a minority of the alpha- syn inside HEK293 cells adopts a highly alpha- helical- rich conformation predominates, indicating that it is preferentially adopted near the cellular periphery. This demonstrates how selectively altering the spatial distriensembles. This approach paves the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell.