Physical Pharmacy Laboratory, Linnaeus University Centre for Biomaterials Chemistry, Linnaeus University, SE-392 31 Kalmar, Sweden.
The Biophysics and Biophotonics Group, Department of Physics, Umeå University, SE-901 87 Umeå, Sweden.
Int J Mol Sci. 2021 Aug 28;22(17):9334. doi: 10.3390/ijms22179334.
Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.
α-突触核蛋白病的特征是脑细胞中有纤维状包涵体。虽然α-突触核蛋白纤维显示出结构多样性，但这种多样性的起源尚未完全了解。我们使用分子动力学模拟设计了基于α-突触核蛋白 NAC 71-82 氨基酸片段的合成肽，这些肽控制原纤维接触和扭曲纤维多态体的生成。选择了四个基于单片段或双片段以及带帽或不带帽末端的结构的肽进行进一步分析。我们通过蛋白浓度测定法和圆二色性光谱法确定了这些肽在纤维形成后的溶液中的纤维产量和结构。此外，我们使用激光镊子拉曼光谱法对单个纤维复合物形成的二级结构进行了表征。结果表明，双片段肽纤维的相对β-折叠含量低于单片段肽纤维，表明形成的纤维较不成熟。我们通过 TEM 分析证实了这种结构差异，该分析显示，在非带帽和带帽的双片段肽系统中，除了短原纤维外，还分别形成了更长、扭曲和棒状的纤维结构。最后，时间相关单光子计数法证明了在纤维结合时，硫黄素 T 荧光寿命分布存在差异。可以提出的是，这种差异源于纤维样品的形态差异。总之，这些结果突出了使用肽模型生成具有与疾病相关的形态特征（例如扭曲和棒状多态体）的纤维的潜力。
