Liu Emilie N, Park Giovanna, Nohara Junsuke, Guo Zhefeng
Department of Neurology, Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
R Soc Open Sci. 2021 Mar 3;8(3):201747. doi: 10.1098/rsos.201747.
Amyloid formation is involved in a wide range of neurodegenerative diseases including Alzheimer's and prion diseases. Structural understanding of the amyloid is critical to delineate the mechanism of aggregation and its pathological spreading. Site-directed spin labelling has emerged as a powerful structural tool in the studies of amyloid structures and provided structural evidence for the parallel in-register β-sheet structure for a wide range of amyloid proteins. It is generally accepted that spin labelling does not disrupt the structure of the amyloid fibrils, the end product of protein aggregation. The effect on the rate of protein aggregation, however, has not been well characterized. Here, we employed a scanning mutagenesis approach to study the effect of spin labelling on the aggregation rate of 79 spin-labelled variants of the Ure2 prion domain. The aggregation of Ure2 protein is the basis of yeast prion [URE3]. We found that all spin-labelled Ure2 mutants aggregated within the experimental timeframe of 15 to 40 h. Among the 79 spin-labelled positions, only five residue sites (N23, N27, S33, I35 and G42) showed a dramatic delay in the aggregation rate as a result of spin labelling. These positions may be important for fibril nucleation, a rate-limiting step in aggregation. Importantly, spin labelling at most of the sites had a muted effect on Ure2 aggregation kinetics, showing a general tolerance of spin labelling in protein aggregation studies.
淀粉样蛋白的形成涉及多种神经退行性疾病，包括阿尔茨海默病和朊病毒疾病。对淀粉样蛋白的结构理解对于阐明聚集机制及其病理传播至关重要。定点自旋标记已成为研究淀粉样蛋白结构的有力结构工具，并为多种淀粉样蛋白的平行同向β-折叠结构提供了结构证据。人们普遍认为，自旋标记不会破坏蛋白质聚集的终产物——淀粉样纤维的结构。然而，其对蛋白质聚集速率的影响尚未得到充分表征。在此，我们采用扫描诱变方法研究自旋标记对Ure2朊病毒结构域79个自旋标记变体聚集速率的影响。Ure2蛋白的聚集是酵母朊病毒[URE3]的基础。我们发现，所有自旋标记的Ure2突变体在15至40小时的实验时间范围内均发生聚集。在79个自旋标记位置中，只有五个残基位点（N23、N27、S33、I35和G42）由于自旋标记而导致聚集速率显著延迟。这些位置可能对纤维成核很重要，而纤维成核是聚集过程中的限速步骤。重要的是，大多数位点的自旋标记对Ure2聚集动力学的影响较小，这表明在蛋白质聚集研究中自旋标记具有普遍耐受性。
