Raman Spectroscopic Studies of Amyloids

拉曼光谱成像是一种强大的技术，它将拉曼光谱的化学特异性与显微镜的空间分辨率相结合。拉曼光谱通常用于测量蛋白质酰胺谱带，其由多肽主链的耦合振动模式产生。酰胺带的位置和宽度取决于肽键角度和氢键模式，因此，通知蛋白质二级结构以及局部环境。 α-螺旋、β-折叠或无规卷曲的构象表现出特征峰最大值，使得结构组成的定量成为可能。 在过去的审查期间，我们完成了两个项目。首先，我们研究了蛋白质的液-液相分离（LLPS）和淀粉样纤维形成的疾病相关过程之间的可能联系。为了确定与淀粉样蛋白形成的机械联系，用拉曼光谱成像的空间分辨率评估液滴内的蛋白质构象状态作为老化时间的函数。在这里，TAR DNA结合蛋白43（TDP-43）的C-末端结构域（CTD），肌萎缩性侧索硬化症相关的蛋白质相分离和形成淀粉样蛋白，进行了研究。使用明场和拉曼光谱成像，直接观察到TDP-43 CTD的液滴成熟，提供了具有空间背景的详细结构信息，否则这些信息将被批量测量所掩盖。最早的聚集事件发生在液滴内，如由β-折叠结构的发展和增加的硫磺素-T发射所指示的。此外，在液滴外部观察到丝状聚集体的较慢出现，此后固化的液滴不与溶液交换。这些结果表明，TDP-43 CTD在相分离条件下的聚集是不均匀的，首先在液滴内发生聚集，然后在溶液中从剩余的单体池形成淀粉样蛋白。此外，液滴内聚集结构的二级结构含量与从头原纤维不同，暗示由于不同的环境（LLPS与本体溶液）而导致的原纤维多态性，这可能具有病理学意义。 在第二个项目中，我们解决了针对其他细胞成分的背景下特异性检测淀粉样纤维的实验挑战。在这里，我们证明了明确识别细胞内化的α-突触核蛋白原纤维的能力，通过耦合拉曼光谱成像与使用的遗传编码的芳基炔，4-乙炔基-L-苯丙氨酸（FCC），通过琥珀密码子抑制。FCC的炔段提供了光谱上独特的分子振动，而不受天然生物分子的干扰。通过拉曼光谱成像，在培养的人SH-SY 5 Y神经母细胞瘤细胞中可视化体外形成的FCC-α-突触核蛋白原纤维的细胞摄取。原纤维表现为不同大小的离散胞质簇，通常在细胞周边发现。内化原纤维的拉曼光谱表现出相对于体外原纤维的频移和光谱变窄，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的脂质和蛋白质募集的变化，并且在某些情况下，观察到原纤维中的二级结构变化。这项工作为未来的拉曼光谱研究奠定了基础，该研究使用类似的方法，即进化的氨酰-tRNA合成酶/tRNA对将FCC掺入内源性淀粉样蛋白中以监测其在细胞中的聚集。

Raman spectral imaging is a powerful technique that couples the chemical specificity of Raman spectroscopy with the spatial resolution of a microscope. Raman spectroscopy is commonly used to measure protein amide bands, which arise from coupled vibrational modes of the polypeptide backbone. The position and widths of the amide bands depend on the peptide-bond angles and hydrogen-bonding patterns, and therefore, inform on protein secondary structure as well as local environment. Conformations of alpha-helix, beta-sheet, or random coil exhibit characteristic peak maxima, making quantification of structural compositions possible. In the past reviewing period, we completed two projects. First, we investigated the possible link between liquid-liquid phase-separation (LLPS) of proteins and the disease-related process of amyloid fibril formation. In order to determine a mechanistic connection to amyloid formation, protein conformation state(s) inside droplets were evaluated as a function of aging time with spatial resolution with Raman spectral imaging. Here, the C-terminal domain (CTD) of TAR DNA-binding protein 43 (TDP-43), an amyotrophic lateral sclerosis-related protein known to both phase-separate and form amyloids, was studied. Using both bright-field and Raman spectral imaging, droplet maturation of TDP-43CTD was directly observed, offering detailed structural information with spatial context which would be otherwise obscured by bulk measurements. The earliest aggregation events occur within droplets as indicated by the development of beta-sheet structure and increased thioflavin-T emission. Additionally, a slower appearance of filamentous aggregates is seen outside the droplets after which time the solidified droplets are not in exchange with the solution. These results suggest that TDP-43CTD aggregation in phase-separating conditions is heterogeneous, with aggregation occurring first within droplets, followed by the formation of amyloids in solution from the remaining pool of monomers. Furthermore, the secondary structure content of aggregated structures inside droplets are distinct from de novo fibrils, implicating fibril polymorphism as a result of the different environments (LLPS vs. bulk solution), which may have pathological significance. In the second project, we addressed the experimental challenge of specific detection of amyloid fibrils against the background of other cellular components. Here, we demonstrate the ability to unambiguously identify cellularly internalized alpha-synuclein fibrils by coupling Raman spectral imaging with the use of a genetically encoded aryl alkyne, 4-ethynyl-L-phenylalanine (FCC), through amber codon suppression. The alkyne stretch of FCC provides a spectrally unique molecular vibration without interference from native biomolecules. Cellular uptake of FCC-alpha-synuclein 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 FCC into endogenous amyloidogenic proteins to monitor their aggregation in cells.