FOSY： intrinsically disordered 蛋白质中的分段骨架分配

中文摘要 AI 辅助

骨架共振分配是大多数生物分子核磁共振（NMR）应用的前提条件，然而，对于 intrinsically disordered 蛋白质（IDPs）和区域（IDRs），传统的多维策略常常因严重的谱峰重叠、酰胺质子与水的快速交换以及顺序相关性的缺失而失败。然而，在许多生物学应用中，完整的蛋白质分配并非必要，因为仅需要围绕功能位点的有限序列片段。在此，我们引入了分段骨架分配（segmental backbone assignment），这是一种由聚焦光谱学（FOcused SpectroscopY，FOSY）实现的分配策略，该策略将实验工作集中在相对较短的区域，同时保留用于明确分配所需的高维顺序连接性。我们提出了一套自洽的选择性二维 FOSY 实验，通过互补的正向和反向转移方案，能够沿蛋白质序列进行双向分配行走。该方法采用频率选择性极化转移，用灵敏且易于解释的二维谱替代高维实验，同时保留多维相关实验的信息内容。该方法的有效性通过完成 G302-K311 片段的分配得到验证，该片段在已发表的 441 个残基的人 Tau 蛋白分配中是缺失的。该方法通过提供一种高效的手段来穿越分配中断并快速表征 intrinsically disordered 蛋白质中功能重要的片段，补充了传统的多维或残基类型选择性分配策略。

英文摘要

Backbone resonance assignment is a prerequisite for most biomolecular NMR applications, yet conventional multidimensional strategies frequently fail for intrinsically disordered proteins (IDPs) and regions (IDRs) because of severe spectral overlap, rapid amide proton exchange with water, and missing sequential correlations. In many biological applications, however, complete protein assignment is unnecessary, as only a limited sequence segment surrounding a functional site is required. Here we introduce segmental backbone assignment, an assignment strategy implemented by FOcused SpectroscopY (FOSY), which concentrates experimental effort on relatively short regions while retaining the high-dimensional sequential connectivity needed for unambiguous assignments. We present a self-consistent suite of selective two-dimensional FOSY experiments that enables bidirectional assignment walks along the protein sequence through complementary forward and backward transfer schemes. The methodology employs frequency-selective polarisation transfer to replace high-dimensional experiments with sensitive and readily interpretable 2D spectra while preserving the information content of multidimensional correlation experiments. The approach is demonstrated by completing the assignment of G302-K311 segment, which is missing in the published assignment of the 441-residue human Tau protein. The approach complements conventional multidimensional or residue type-selective assignment strategies by providing an efficient means of traversing assignment interruptions and rapidly characterising functionally important segments in intrinsically disordered proteins.

发表机构

• University of Gothenburg（哥德堡大学）
• Shemyakin-Ovchinnikov, Institute of Bioorganic Chemistry RAS（谢米亚金-奥夫钦尼科夫俄罗斯科学院生物有机化学研究所）
• Swedish NMR Centre and SciLifeLab, University of Gothenburg（哥德堡大学瑞典核磁共振中心与科学生命实验室）
• Swedish University of Agricultural Sciences（瑞典农业科学大学）
• Department of Psychiatry and Neurochemistry, Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg（哥德堡大学瓦伦伯格分子与转化医学中心精神病学与神经化学系）
• Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg（哥德堡大学瓦伦伯格分子与转化医学中心）

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