Department of Inorganic Chemistry, University of Granada, 18071 Granada, Spain.
Department of Health Sciences and Technology, ETH Zürich, 8092 Zürich, Switzerland.
Biomacromolecules. 2021 May 10;22(5):2057-2066. doi: 10.1021/acs.biomac.1c00176. Epub 2021 Apr 6.
We present the optimization of experimental conditions to yield long, rigid apoferritin protein amyloid fibrils, as well as the corresponding fibrillation pathway. Fibril growth kinetics was followed using atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS), circular dichroism (CD), fourier-transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among the morphologies identified, we show that the conditions result in small aggregates, as well as medium and long fibrils. Extended incubation times led to progressive unfolding and hydrolysis of the proteins into very short peptide fragments. AFM, SDS-PAGE, and CD support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role. These collective results provide convincing evidence that protein unfolding and complete hydrolysis of the proteins into very short peptide sequences are essential for the formation of the final apoferritin amyloid-like fibrils.
我们提出了优化实验条件以获得长而刚性的脱铁蛋白蛋白淀粉样纤维的方法，以及相应的纤维化途径。使用原子力显微镜（AFM）、透射电子显微镜（TEM）、动态光散射（DLS）、圆二色性（CD）、傅里叶变换红外光谱（FTIR）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）跟踪纤维生长动力学。在所确定的形态中，我们表明，这些条件导致小聚集体以及中长纤维的形成。延长孵育时间会导致蛋白质逐渐展开并水解成非常短的肽片段。AFM、SDS-PAGE 和 CD 支持普遍的共同纤维化机制，其中水解片段起着核心作用。这些综合结果提供了令人信服的证据，表明蛋白质展开和蛋白质完全水解成非常短的肽序列对于最终形成脱铁蛋白样淀粉样纤维是必不可少的。
