Synchrotron SOLEIL, L'Orme des Merisiers Saint Aubin, Gif-sur-Yvette, France.
Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay, CEA Saclay, Gif-sur-Yvette, France.
J Microsc. 2019 Apr;274(1):23-31. doi: 10.1111/jmi.12779. Epub 2019 Jan 16.
Propagation of structural information through conformational changes in host-encoded amyloid proteins is at the root of many neurodegenerative disorders. Although important breakthroughs have been made in the field, fundamental issues like the 3D-structures of the fibrils involved in some of those disorders are still to be elucidated. To better characterise those nanometric fibrils, a broad range of techniques is currently available. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of the C-terminal region of a bacterial protein called Hfq as a model amyloidogenic protein, using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM-IR. We introduce and discuss the strategy that we have implemented as well as the protocol, challenges and difficulties encountered during this study to characterise amyloid assemblies at the nearly single-molecule level. LAY DESCRIPTION: Propagation of structural information through conformational changes in amyloid proteins is at the root of many neurodegenerative disorders. Amyloids are nanostructures originating from the aggregation of multiple copies of peptide or protein monomers that eventually form fibrils. Often described as being the cause for the development of various diseases, amyloid fibrils are of major significance in the public health domain. While important breakthroughs have been made in the field, fundamental issues like the 3D-structures of the fibrils implied in some of those disorders are still to be elucidated. To better characterise these fibrils, a broad range of techniques is currently available for the detection and visualisation of amyloid nanostructures. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of model amyloidogenic fibrils using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM-IR that allows chemical characterisation at the nanometric scale. The strategy, protocol, challenges and difficulties encountered in this approach are introduced and discussed herein.
结构信息通过宿主编码淀粉样蛋白的构象变化传播是许多神经退行性疾病的根源。尽管该领域取得了重要突破，但仍有一些基本问题尚未解决，例如涉及其中一些疾病的纤维的 3D 结构。为了更好地描述这些纳米纤维，目前有广泛的技术可供使用。然而，没有一种技术能够对单个蛋白质纤维进行直接的化学表征。在这项工作中，我们提议使用相关方法研究一种称为 Hfq 的细菌蛋白的 C 末端区域作为模型淀粉样蛋白的结构。使用的互补技术是透射电子显微镜和一种新开发的称为 AFM-IR 的红外纳米光谱技术。我们介绍并讨论了我们实施的策略以及在这项研究中遇到的方案、挑战和困难，以在近单分子水平上表征淀粉样组装体。
结构信息通过淀粉样蛋白的构象变化传播是许多神经退行性疾病的根源。淀粉样蛋白是源自多个肽或蛋白质单体聚合的纳米结构，最终形成纤维。淀粉样纤维通常被描述为导致各种疾病发展的原因，在公共卫生领域具有重要意义。尽管该领域取得了重要突破，但仍有一些基本问题尚未解决，例如涉及其中一些疾病的纤维的 3D 结构。为了更好地描述这些纤维，目前有广泛的技术可用于检测和可视化淀粉样纳米结构。然而，没有一种技术能够对单个蛋白质纤维进行直接的化学表征。在这项工作中，我们提议使用相关方法研究模型淀粉样蛋白纤维的结构。使用的互补技术是透射电子显微镜和一种新开发的红外纳米光谱技术，称为 AFM-IR，它允许在纳米尺度上进行化学表征。本文介绍并讨论了该方法中的策略、方案、挑战和困难。
