Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
Photonics Center, Boston University, Boston, MA 02215, USA.
Angew Chem Int Ed Engl. 2024 Aug 26;63(35):e202408163. doi: 10.1002/anie.202408163. Epub 2024 Jul 22.
While protein aggregation is a hallmark of many neurodegenerative diseases, acquiring structural information on protein aggregates inside live cells remains challenging. Traditional microscopy does not provide structural information on protein systems. Routinely used fluorescent protein tags, such as Green Fluorescent Protein (GFP), might perturb native structures. Here, we report a counter-propagating mid-infrared photothermal imaging approach enabling mapping of secondary structure of protein aggregates in live cells modeling Huntington's disease. By comparing mid-infrared photothermal spectra of label-free and GFP-tagged huntingtin inclusions, we demonstrate that GFP fusions indeed perturb the secondary structure of aggregates. By implementing spectra with small spatial step for dissecting spectral features within sub-micrometer distances, we reveal that huntingtin inclusions partition into a β-sheet-rich core and a ɑ-helix-rich shell. We further demonstrate that this structural partition exists only in cells with the [RNQ] prion state, while [rnq] cells only carry smaller β-rich non-toxic aggregates. Collectively, our methodology has the potential to unveil detailed structural information on protein assemblies in live cells, enabling high-throughput structural screenings of macromolecular assemblies.
虽然蛋白质聚集是许多神经退行性疾病的标志，但在活细胞内获取蛋白质聚集体的结构信息仍然具有挑战性。传统显微镜无法提供蛋白质系统的结构信息。常规使用的荧光蛋白标签，如绿色荧光蛋白（GFP），可能会干扰天然结构。在这里，我们报告了一种反向传播的中红外光热成像方法，可用于对亨廷顿病模型活细胞中的蛋白质聚集体的二级结构进行成像。通过比较无标记和 GFP 标记的亨廷顿蛋白包涵体的中红外光热光谱，我们证明 GFP 融合确实会干扰聚集体的二级结构。通过实施具有小空间步长的光谱来解析亚微米距离内的光谱特征，我们揭示亨廷顿包涵体分为富含β-折叠的核心和富含α-螺旋的外壳。我们进一步证明，这种结构分区仅存在于具有 [RNQ] 朊病毒状态的细胞中，而 [rnq] 细胞仅携带较小的β-丰富非毒性聚集体。总的来说，我们的方法有可能揭示活细胞中蛋白质组装的详细结构信息，从而实现对大分子组装的高通量结构筛选。
