Watching liquid droplets of TDP-43CTD age by Raman spectroscopy

Abstract

Liquid–liquid phase separation (LLPS) is a biological phenomenon wherein a metastable and concentrated droplet phase of biomolecules spontaneously forms. A link may exist between LLPS of proteins and the disease-related process of amyloid fibril formation; however, this connection is not fully understood. Here, we investigated the relationship between LLPS and aggregation of the C-terminal domain of TAR DNA-binding protein 43, an amyotrophic lateral sclerosis–related protein known to both phase separate and form amyloids, by monitoring conformational changes during droplet aging using Raman spectroscopy. We found that the earliest aggregation events occurred within droplets as indicated by the development of β-sheet structure and increased thioflavin-T emission. Interestingly, filamentous aggregates appeared outside the solidified droplets at a later time, suggestive that amyloid formation is a heterogeneous process under LLPS solution conditions. Furthermore, the secondary structure content of aggregated structures inside droplets is distinct from that in de novo fibrils, implying that fibril polymorphism develops as a result of different environments (LLPS versus bulk solution), which may have pathological significance.

Micro-Raman spectroscopic analysis of liquid–liquid phase separation

“…In particular, for proteins in a fibril state, the β-sheet characteristic increases in the amide I band region, which becomes narrower and is located at ∼1671 cm −1 . 28,42,55 Notably, Fig. 3(c) shows that the peak position of the amide I band remained unchanged (∼1655 cm −1 ), and there was also little difference in the linewidth between the concentrated protein solution and the inside droplets.…”

“…Micro-Raman spectroscopy is a promising approach to avoid or ameliorate these problems. 23,27,28 In particular, micro-Raman spectroscopy has several advantages for analyzing LLPS processes, allowing label-free, non-destructive measurement and structural analysis of the components inside and outside liquid droplets at a suitable spatial resolution. First, the micro-Raman spectrometer is commercially available.…”

Concentration Quantification of the Low-Complexity Domain of Fused in Sarcoma inside a Single Droplet and Effects of Solution Parameters

“…Raman microscopy has recently been studied as a label-free technique for observing biological systems. − In a previous study, we proposed that Raman imaging can be applied to measure protein concentration inside a liquid droplet under single-droplet and label-free conditions. There have been several reports on LLPS analysis using Raman microscopy, − and our proposed method focuses on the fact that the concentration of water outside droplets is extremely high, and thus, the water Raman band can be used as an internal intensity standard for quantifying the protein Raman bands. We used the O–H stretching Raman band of water, which is strongly observed in aqueous solutions, as an intensity standard .…”

Single-Droplet Surface-Enhanced Raman Scattering Decodes the Molecular Language of Liquid-Liquid Phase Separation

“…Vibrational Raman spectroscopy performed in a microscopy format allows us to uniquely and elegantly combine the aforesaid capabilities to obtain the protein structural information from a well-defined spatial location by focusing the laser beam into a sub-micron spot. Such non-invasive and label-free laser micro-Raman measurements permit us to access the wealth of structural information by monitoring a range of bond vibrational frequencies while retaining the spatial resolution 24,25,26 . However, owing to a low Raman scattering cross-section, Raman spectroscopy is a highly insensitive technique, especially for biomolecules under physiological conditions in aqueous solutions 27 .…”