Intze Antonia, Temperini Maria Eleonora, Rupert Jakob, Polito Raffaella, Veber Alexander, Puskar Ljiljana, Schade Ulrich, Ortolani Michele, Zacco Elsa, Tartaglia Gian Gaetano, Giliberti Valeria
Center for Life Nano- & Neuro-science, Istituto Italiano di Tecnologia (IIT), Rome, Italy; Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, Rome, Italy; Department of Physics, Sapienza University of Rome, Rome, Italy.
Center for Life Nano- & Neuro-science, Istituto Italiano di Tecnologia (IIT), Rome, Italy; Department of Physics, Sapienza University of Rome, Rome, Italy.
Biophys J. 2025 Jun 17;124(12):2005-2019. doi: 10.1016/j.bpj.2025.04.031. Epub 2025 May 5.
Structural changes associated with protein aggregation are challenging to study, requiring the combination of experimental techniques providing insights at the molecular level across diverse scales, ranging from nanometers to microns. Understanding these changes is even more complex when aggregation occurs in the presence of molecular cofactors such as nucleic acids and when the resulting aggregates are highly polymorphic. Infrared (IR) spectroscopy is a powerful tool for studying protein aggregates since it combines the label-free sensitivity to the cross-β architecture, an inherent feature of protein supramolecular aggregates, with the possibility to reach nanoscale sensitivity by leveraging atomic force microscopy (AFM)-assisted detection. Here, we present a combined approach that detects IR spectral markers of aggregation using various IR spectroscopy techniques, covering micro-to-nanoscale ranges, to study the effect of RNA on the supramolecular architecture of α-synuclein amyloid aggregates. We show a clear impact of RNA consistent with enhanced intermolecular forces, likely via a stronger hydrogen-bonded network stabilizing the cross-β architecture. AFM-assisted IR spectroscopy was crucial to assess that the more ordered the aggregates are, the stronger the structural impact of RNA. In addition, an RNA-induced reduction of the degree of polymorphism within the aggregate population is obtained.
与蛋白质聚集相关的结构变化很难研究，需要结合多种实验技术，这些技术要能在从纳米到微米的不同尺度上提供分子水平的见解。当聚集在诸如核酸等分子辅助因子存在的情况下发生，且所形成的聚集体具有高度多态性时，理解这些变化就更加复杂。红外（IR）光谱是研究蛋白质聚集体的有力工具，因为它将对交叉β结构（蛋白质超分子聚集体的固有特征）的无标记敏感性，与通过利用原子力显微镜（AFM）辅助检测达到纳米级敏感性的可能性结合起来。在此，我们提出一种组合方法，使用各种红外光谱技术（涵盖微米到纳米尺度范围）检测聚集的红外光谱标记，以研究RNA对α-突触核蛋白淀粉样聚集体超分子结构的影响。我们展示了RNA的明显影响，这与增强的分子间作用力一致，可能是通过更强的氢键网络稳定交叉β结构实现的。AFM辅助红外光谱对于评估聚集体越有序，RNA的结构影响就越强至关重要。此外，还观察到RNA诱导聚集体群体内多态性程度降低。
