<br>使用单分子取向定位显微镜解析 β 片肽自组装的纳米级结构,ACS Nano

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使用单分子取向定位显微镜解析 β 片肽自组装的纳米级结构
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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由于其模块化和生物相容性，自组装成交叉β原纤维的合成肽是工程生物材料的多功能构建单元，但它们与淀粉样蛋白物种的结构和形态相似性一直是其转化的长期关注点。此外，它们的多晶型物很难通过使用依赖于集成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），一种亲淀粉样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异构体 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，并在相关缓冲条件下量化了原纤维内骨架和外骨架的精确倾斜，而无需共价标记或序列突变。SMOLM 还区分了 KFE8 的多态性分支和弯曲形态，其骨架比典型的直原纤维表现出更多的异质性。因此，SMOLM 是询问工程和病理交叉β富原纤维之间结构差异和多态性的有力工具。

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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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