通过使用界面活性溶液和观众蛋白来改善粉样纤维的冷-EM网格
Dylan Valli1, Saik Ann Ooi2, Giorgio Scattolini1
1Department of Chemistry - Ångström Laboratory, Uppsala University, Uppsala, Sweden.
Biophysical journal
|February 18, 2024
概括
研究人员开发了新的电子显微镜 (cryo-EM) 网格准备方法,用于粉样纤维. HEPES缓冲器和"观众蛋白"改善了纤维分布和冰质量,使人类小岛粉样多 (hIAPP) 的结构确定成为可能.
科学领域:
- 生物物理学的生物物理.
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- 准备冷电子显微镜 (cryo-EM) 网格用于粉样纤维素成像,例如人类小岛粉样多 (hIAPP),具有挑战性.
- 现有的方法通常需要非生理条件,标签或播种,限制了在不同条件下形成的纤维的结构研究.
- 有效和可重复的网格准备对于阐明粉样纤维结构至关重要.
研究的目的:
- 为粉样纤维素开发改进的冷电子显微镜 (cryo-EM) 网格制备技术.
- 为了识别可增强纤维分布和冰稳定的溶液成分,用于成像.
- 为了确定在生理学pH下体外形成的主导hIAPP多态的结构.
主要方法:
- 对缓冲组合的系统评估,重点是生理pH,用于hIAPP的冷-EM电网准备.
- 研究溶液成分在稳定薄玻璃冰膜中的作用.
- 使用非粉原性大鼠IAPP (rIAPP) 作为"观众蛋白"来提高颗粒覆盖率和冰质量.
主要成果:
- 鉴定出HEPES缓冲剂在生理pH下独特地增强了hIAPP纤维分布和冰层稳定性.
- HEPES与hIAPP直接相互作用,减少聚合并防止冰核形成.
- 添加rIAPP改善了电网覆盖率和冰质量,作为表面活性剂并解开hIAPP集群.
- 在pH 7.4时,占主导地位的hIAPP多态的结构被分解为4 Å分辨率.
结论:
- 溶液的组成极大地影响了纤维的分布和冰的稳定性在冷-EM网格的准备.
- HEPES缓冲器和rIAPP的添加代表了在制备粉样纤维网格方面取得的重大进展.
- 这些策略使得以前对常规方法耐药的粉样蛋白的结构确定成为可能.
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