Site-Specific Raman Probes Reveal Droplet Aging and Residue-Level Fibril Polymorphism in TDP-43_CTD

The C-terminal domain of TAR DNA-binding protein 43 (TDP-43_CTD) drives both liquid-liquid phase separation (LLPS) and amyloid formation. Understanding how TDP-43_CTD droplets convert into amyloid aggregates, a process implicated in amyotrophic lateral sclerosis and frontotemporal dementia, requires methodology capable of site-specific structural characterization with spatial resolution. Here, we used confocal Raman spectroscopy in conjunction with an alkyne-modified amino acid (4-ethynyl-l-phenylalanine, F_CC) to probe aging in individual TDP-43_CTD droplets at seven aromatic sites. While nascent droplets are composed of disordered proteins, β-sheet conformers develop in aged droplets and amyloid aggregates. All three states are spectrally distinct via the alkyne stretching band, with sensitivity that varies depending on the aromatic site probed. C-terminal sites (Y374F_CC, W385F_CC, and F397F_CC) are highly sensitive amyloid probes, revealing multiple polymorphs at the single-residue level that are not resolvable by global secondary structure or morphological characterization alone. Strikingly, while W334F_CC abolishes β-sheet formation in droplets, de novo aggregation still occurs, demonstrating that droplet aging is not required for amyloid formation. Given its broad applicability to other proteins and compatibility with cellular imaging, this work establishes a generalizable approach for investigating conformational changes underlying LLPS and amyloid formation in cellulo.