Laboratory of Protein Conformation and Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, United States.
Laboratory of Protein Conformation and Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, United States.
J Mol Biol. 2023 Jan 15;435(1):167716. doi: 10.1016/j.jmb.2022.167716. Epub 2022 Jul 2.
α-Synuclein (α-syn) is an intrinsically disordered protein involved in a group of diseases collectively termed synucleinopathies, characterized by the aggregation of α-syn to form insoluble, β-sheet-rich amyloid fibrils. Amyloid fibrils are thought to contribute to disease progression through cell-to-cell transmission, templating and propagating intracellular amyloid formation. Raman spectral imaging offers a direct characterization of protein secondary structure via the amide-I backbone vibration; however, specific detection of α-syn conformational changes against the background of other cellular components presents a challenge. Here, we demonstrate the ability to unambiguously identify cellularly internalized α-syn fibrils by coupling Raman spectral imaging with the use of a genetically encoded aryl alkyne, 4-ethynyl-l-phenylalanine (F), through amber codon suppression. The alkyne stretch (CC) of F provides a spectrally unique molecular vibration without interference from native biomolecules. Cellular uptake of F-α-syn fibrils formed in vitro was visualized in cultured human SH-SY5Y neuroblastoma cells by Raman spectral imaging. Fibrils appear as discrete cytosolic clusters of varying sizes, found often at the cellular periphery. Raman spectra of internalized fibrils exhibit frequency shifts and spectral narrowing relative to in vitro fibrils, highlighting the environmental sensitivity of the alkyne vibration. Interestingly, spectral analysis reveals variations in lipid and protein recruitment to these aggregates, and in some cases, secondary structural changes in the fibrils are observed. This work sets the groundwork for future Raman spectroscopic investigations using a similar approach of an evolved aminoacyl-tRNA synthetase/tRNA pair to incorporate F into endogenous amyloidogenic proteins to monitor their aggregation in cells.
α-突触核蛋白（α-syn）是一种无规则卷曲的蛋白质，涉及一类统称为突触核蛋白病的疾病，其特征是α-syn 聚集形成不溶性、β-折叠丰富的淀粉样纤维。淀粉样纤维被认为通过细胞间传递、模板和传播细胞内淀粉样形成来促进疾病进展。拉曼光谱成像通过酰胺-I 骨架振动提供蛋白质二级结构的直接特征化；然而，在其他细胞成分的背景下，对 α-syn 构象变化的特异性检测提出了挑战。在这里，我们通过将拉曼光谱成像与使用通过琥珀密码子抑制遗传编码的芳基炔烃 4-乙炔基-l-苯丙氨酸（F）相结合，证明了能够明确识别细胞内内化的α-syn 纤维的能力。F 的炔伸展（CC）提供了光谱独特的分子振动，而不会受到天然生物分子的干扰。通过拉曼光谱成像，在培养的人 SH-SY5Y 神经母细胞瘤细胞中可视化了体外形成的 F-α-syn 纤维的细胞摄取。纤维呈现为不同大小的离散细胞溶质簇，通常位于细胞边缘。与体外纤维相比，内化纤维的拉曼光谱显示出频率位移和光谱变窄，突出了炔振动的环境敏感性。有趣的是，光谱分析揭示了这些聚集体中脂质和蛋白质募集的变化，并且在某些情况下观察到纤维的二级结构变化。这项工作为未来使用类似的方法，即进化的氨酰-tRNA 合成酶/tRNA 对将 F 掺入内源性淀粉样蛋白原蛋白中以监测其在细胞中的聚集的拉曼光谱研究奠定了基础。
