Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
Faculty of Mathematics and Physics, University of Ljubljana, Jadranska 19, 1000 Ljubljana, Slovenia.
Int J Mol Sci. 2023 Feb 13;24(4):3737. doi: 10.3390/ijms24043737.
Human stefin B, a member of the cystatin family of cysteine protease inhibitors, tends to form amyloid fibrils under relatively mild conditions, which is why it is used as a model protein to study amyloid fibrillation. Here, we show for the first time that bundles of amyloid fibrils, i.e., helically twisted ribbons, formed by human stefin B exhibit birefringence. This physical property is commonly observed in amyloid fibrils when stained with Congo red. However, we show that the fibrils arrange in regular anisotropic arrays and no staining is required. They share this property with anisotropic protein crystals, structured protein arrays such as tubulin and myosin, and other anisotropic elongated materials, such as textile fibres and liquid crystals. In certain macroscopic arrangements of amyloid fibrils, not only birefringence is observed, but also enhanced emission of intrinsic fluorescence, implying a possibility to detect amyloid fibrils with no labels by using optical microscopy. In our case, no enhancement of intrinsic tyrosine fluorescence was observed at 303 nm; instead, an additional fluorescence emission peak appeared at 425 to 430 nm. We believe that both phenomena, birefringence and fluorescence emission in the deep blue, should be further explored with this and other amyloidogenic proteins. This may allow the development of label-free detection methods for amyloid fibrils of different origins.
人源组织蛋白酶抑制剂 B（Stefin B）属于半胱氨酸蛋白酶抑制剂家族成员，在相对温和的条件下易于形成淀粉样纤维，因此常被用作研究淀粉样纤维形成的模型蛋白。在此，我们首次证明由人源组织蛋白酶抑制剂 B 形成的淀粉样纤维束（即螺旋扭曲的带状物）具有双折射性。这一物理性质在使用刚果红染色的淀粉样纤维中很常见。然而，我们发现纤维呈规则各向异性排列，无需染色。它们与各向异性蛋白晶体、微管蛋白和肌球蛋白等结构化蛋白阵列以及其他各向异性细长材料（如纺织纤维和液晶）具有相同的性质。在某些淀粉样纤维的宏观排列中，不仅观察到双折射，还观察到固有荧光的增强发射，这意味着可以通过光学显微镜检测无标记的淀粉样纤维。在我们的情况下，在 303nm 处未观察到酪氨酸固有荧光的增强；相反，在 425 到 430nm 处出现了一个额外的荧光发射峰。我们相信，双折射和深蓝光下的荧光发射这两种现象都应该用这种和其他淀粉样蛋白进一步探索。这可能会为不同来源的淀粉样纤维开发无标记检测方法。
