Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, 412 96 Gothenburg, Sweden.
Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, 421 Washington Ave SE, Minneapolis, Minnesota 55455, USA.
Nanoscale. 2023 Nov 30;15(46):18737-18744. doi: 10.1039/d3nr02740f.
Amyloid fibril formation is central to the pathology of many diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Amyloid fibrils can also have functional and scaffolding roles, for example in bacterial biofilms, and have also been exploited as useful biomaterials. Despite being linear protein homopolymers, amyloid fibrils can exhibit significant structural and morphological polymorphism, making it relevant to study them on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently-labeled amyloid fibrils in solution, monitoring the extension and emission intensity of individual fibrils confined in nanochannels with a depth of 300 nm and a width that gradually increases from 300 to 3000 nm. The change in fibril extension with channel width permitted accurate determination of the persistence length of individual fibrils using Odijk's theory for strongly confined polymers. The technique was applied to amyloid fibrils prepared from the Alzheimer's related peptide amyloid-β(1-42) and the Parkinson's related protein α-synuclein, obtaining mean persistence lengths of 5.9 ± 4.5 μm and 3.0 ± 1.6 μm, respectively. The broad distributions of fibril persistence lengths indicate that amyloid fibril polymorphism can manifest in their physical properties. Interestingly, the α-synuclein fibrils had lower persistence lengths than the amyloid-β(1-42) fibrils, despite being thicker. Furthermore, there was no obvious within-sample correlation between the fluorescence emission intensity per unit length of the labelled fibrils and their persistence lengths, suggesting that stiffness may not be proportional to thickness. We foresee that the nanofluidics methodology established here will be a useful tool to study amyloid fibrils on the single fibril level to gain information on heterogeneity in their physical properties and interactions.
淀粉样纤维的形成是许多疾病的病理学核心，包括神经退行性疾病，如阿尔茨海默病和帕金森病。淀粉样纤维也具有功能和支架作用，例如在细菌生物膜中，并且也被用作有用的生物材料。尽管是线性蛋白质均聚物，但淀粉样纤维可以表现出显著的结构和形态多态性，因此在单纤维水平上研究它们是相关的。我们在这里将纳米流体通道分析的概念引入到溶液中单个荧光标记淀粉样纤维的研究中，监测在深度为 300nm 且宽度逐渐从 300nm 增加到 3000nm 的纳米通道中限制的单个纤维的延伸和发射强度。纤维延伸与通道宽度的变化允许使用 Odijk 理论对强限制聚合物的单个纤维的持久长度进行准确确定。该技术应用于从阿尔茨海默病相关肽淀粉样蛋白-β(1-42)和帕金森病相关蛋白α-突触核蛋白制备的淀粉样纤维，得到的单个纤维的平均持久长度分别为 5.9±4.5μm 和 3.0±1.6μm。纤维持久长度的广泛分布表明，淀粉样纤维的多态性可以表现在它们的物理性质上。有趣的是，尽管α-突触核蛋白纤维更厚，但它们的持久长度比淀粉样蛋白-β(1-42)纤维低。此外，标记纤维的单位长度荧光发射强度与其持久长度之间没有明显的样本内相关性，这表明刚性可能与厚度不成比例。我们预计，这里建立的纳米流体学方法将成为研究单纤维淀粉样纤维的有用工具，以获得有关其物理性质和相互作用异质性的信息。
