Shuster Sydney O, Lee Jennifer C
Laboratory of Protein Conformation and Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
J Phys Chem B. 2021 Apr 22;125(15):3781-3789. doi: 10.1021/acs.jpcb.1c00767. Epub 2021 Apr 9.
Aggregated TAR DNA-binding protein 43 (TDP-43) forms the cytoplasmic hallmarks associated with patients suffering from amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin. Under normal conditions, TDP-43 is a 414-amino acid protein; however, aggregates are enriched with N-terminal truncations which contain residues 267-414, known as the C-terminal domain of TDP-43 (TDP-43). To gain residue-specific information on the aggregation process of TDP-43, we created three single-Trp containing mutants (W385F/W412F, W334F/W412F, and W334F/W385F) by substituting two of the three native Trp residues with Phe, yielding fluorescent probes at W334, W385, and W412, respectively. Aggregation kinetics, secondary structure, and fibril morphology were compared to the wild-type protein using thioflavin-T fluorescence, Raman spectroscopy, and transmission electron microscopy, respectively. While only W334 is determined to be in the proteinase-K resistant core, all three sites are sensitive reporters of aggregation, revealing site-specific differences. Interestingly, W334 exhibited unusual multistep Trp kinetics, pinpointing a distinctive role for W334 and its nearby region during aggregation. This behavior is retained even upon seeding, suggesting the observed spectral change is related to fibril growth. This work provides new insights into the aggregation mechanism of TDP-43 and exemplifies the advantages of Trp as a site-specific environmentally sensitive fluorescent probe.
聚集的TAR DNA结合蛋白43（TDP - 43）形成与肌萎缩侧索硬化症和伴有泛素的额颞叶痴呆症患者相关的细胞质特征。在正常情况下，TDP - 43是一种由414个氨基酸组成的蛋白质；然而，聚集体富含N端截短产物，其包含267 - 414位残基，即TDP - 43的C端结构域（TDP - 43）。为了获得关于TDP - 43聚集过程的残基特异性信息，我们通过用苯丙氨酸取代三个天然色氨酸残基中的两个，创建了三个含单个色氨酸的突变体（W385F/W412F、W334F/W412F和W334F/W385F），分别在W334、W385和W412处产生荧光探针。分别使用硫黄素 - T荧光、拉曼光谱和透射电子显微镜将聚集动力学、二级结构和原纤维形态与野生型蛋白进行比较。虽然仅确定W334位于蛋白酶K抗性核心中，但所有三个位点都是聚集的敏感报告分子，揭示了位点特异性差异。有趣的是，W334表现出不寻常的多步色氨酸动力学，指出了W334及其附近区域在聚集过程中的独特作用。即使在接种后这种行为仍然保留，表明观察到的光谱变化与原纤维生长有关。这项工作为TDP - 43的聚集机制提供了新的见解，并例证了色氨酸作为位点特异性环境敏感荧光探针的优势。
