Dey Arpan, Maiti Sudipta
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India.
Methods Mol Biol. 2022;2538:55-74. doi: 10.1007/978-1-0716-2529-3_5.
Small oligomers are the initial intermediates in the pathway to amyloid fibril formation. They have a distinct identity from the monomers as well as from the protofibrils and the fibrils, both in their structure and in their properties. In many cases, they play a crucial biological role. However, due to their transient nature, they are difficult to characterize. "Oligomer" is a diffuse definition, encompassing aggregates of many different sizes, and this lack of precise definition causes much confusion and disagreement between different research groups. Here, we define the small oligomers as "n"-mers with n < 10, which is the size range in which the amyloid proteins typically exist at the initial phase of the aggregation process. Since the oligomers dynamically interconvert into each other, a solution of aggregating amyloid proteins will contain a distribution of sizes. A precise characterization of an oligomeric solution will, therefore, require quantification of the relative population of each size. Size-based separation methods, such as size-exclusion chromatography, are typically used to characterize this distribution. However, if the interconversion between oligomers of different sizes is fast, this would not yield reliable results. Single-molecule photobleaching (smPB) is a direct method to evaluate this size distribution in a heterogeneous solution without separation. In addition, understanding the mechanism of action of amyloid oligomers requires knowing the affinity of each oligomer type to different cellular components, such as the cell membrane. These measurements are also amenable to smPB. Here we show how to perform smPB, both for oligomers in solution and for oligomers attached to the membrane.
小分子寡聚体是淀粉样纤维形成途径中的初始中间体。它们在结构和性质上与单体、原纤维和纤维都有明显的区别。在许多情况下，它们发挥着关键的生物学作用。然而，由于其短暂性，它们很难被表征。“寡聚体”是一个宽泛的定义，涵盖了许多不同大小的聚集体，这种缺乏精确的定义导致了不同研究小组之间的许多困惑和分歧。在这里，我们将小分子寡聚体定义为“n”聚体，其中n < 10，这是淀粉样蛋白在聚集过程初始阶段通常存在的大小范围。由于寡聚体之间会动态相互转化，聚集的淀粉样蛋白溶液将包含不同大小的分布。因此，对寡聚体溶液进行精确表征需要对每种大小的相对数量进行量化。基于大小的分离方法，如尺寸排阻色谱法，通常用于表征这种分布。然而，如果不同大小的寡聚体之间的相互转化很快，这将不会产生可靠的结果。单分子光漂白（smPB）是一种在不分离的情况下评估异质溶液中这种大小分布的直接方法。此外，了解淀粉样寡聚体的作用机制需要知道每种寡聚体类型对不同细胞成分（如细胞膜）的亲和力。这些测量也适用于smPB。在这里，我们展示了如何对溶液中的寡聚体和附着在膜上的寡聚体进行smPB。
