<br>使用单分子定向定位显微镜解析 β-折叠肽自组装体的纳米级结构,ACS Nano

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使用单分子定向定位显微镜解析 β-折叠肽自组装体的纳米级结构
ACS Nano ( IF 15.8 ) Pub Date : 2024-03-13 , DOI: 10.1021/acsnano.3c11771
Weiyan Zhou ₁ , Conor L O'Neill ₂ , Tianben Ding ₁ , Oumeng Zhang ₁ , Jai S Rudra ₂ , Matthew D Lew ₁

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自组装成交叉β原纤维的合成肽由于其模块化和生物相容性而成为工程生物材料的通用构建模块，但它们与淀粉样蛋白物种的结构和形态相似性一直是其翻译的长期关注点。此外，它们的多晶型物很难通过使用依赖于整体平均来实现高分辨率的光谱和成像技术来表征。在这里，我们利用尼罗红（NR）（一种淀粉样蛋白荧光探针）和单分子定向定位显微镜（SMOLM）来表征由设计的两亲性对映体 KFE8 ^L和 KFE8 ^D以及病理性淀粉样蛋白-β 肽 Aβ42 形成的原纤维。重要的是，NR SMOLM 揭示了 KFE8 和 Aβ42 的螺旋（双层）带状结构，并量化了相关缓冲条件下原纤维内部和外部主链的精确倾斜，无需共价标记或序列突变。 SMOLM 还区分了 KFE8 的多态分支和弯曲形态，其主链比典型的直原纤维表现出更多的异质性。因此，SMOLM 是一种强大的工具，可用于研究工程和病理性富含交叉β原纤维之间的结构差异和多态性。

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Resolving the Nanoscale Structure of β-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation–Localization Microscopy

Synthetic peptides that self-assemble into cross-β fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation–localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8^L and KFE8^D and the pathological amyloid-beta peptide Aβ42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aβ42 and quantifies the precise tilt of the fibrils’ inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-β-rich fibrils.

更新日期：2024-03-13

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