Project Summary The TAR DNA-binding protein of 43 kDa (TDP-43) is a nucleic-acid binding protein whose fragments have been detected in pathological amyloid inclusions in the brains of patients with age-related neurodegenerative disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration. These fragments contain a region of TDP-43 called the low complexity domain (LCD), which has low amino acid diversity and a propensity to form amyloid aggregates. The LCD also drives TDP-43 liquid-liquid phase separation (LLPS), a phenomenon in which the protein self-associates into a reversible, dynamic, droplet-like phase. LLPS is a physiologic process that mediates several functions of TDP-43, including its involvement in the cellular stress response, but prolonged LLPS has been shown to promote pathological aggregation, underscoring the need to understand how this process is regulated in vivo. Phosphorylation of TDP-43 LCD has been well-documented in Alzheimer’s disease and other age-related neurodegenerative disorders. Though the role of this modification remains largely unexplored for TDP-43, it does influence LLPS in similar RNA-binding proteins such as FUS. To determine if LCD phosphorylation can regulate TDP-43 LLPS, phosphomimetic variants of the LCD with Ser->Asp substitutions at known phosphorylation sites were prepared. Phase separation of variants was studied using light-scattering and microscopy, and it was found the phosphomimetic substitutions dramatically modulated LLPS in a site-specific manner. LCD variants with phosphomimetic substitutions in the α-helical region, a part of the protein involved in protein-protein contacts, displayed reduced LLPS-propensity; conversely, LCD variants with phosphomimetic substitutions in the C- terminal region (CTR), which is known to be phosphorylated in disease, displayed enhanced LLPS-propensity. To elucidate the mechanisms behind this site-specific modulation of LLPS, we propose a three-pronged approach using nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and fluorescence resonance energy transfer (FRET). NMR will be employed to determine local secondary structure of the phosphomimetic α-helical variant. As the α-helical region is known to be altered by oligomerization, EPR will be used concurrently to see if the α-helical phosphomimetic substitution changes the oligomerization behavior of this variant. Preliminary data suggest the CTR variants have higher LLPS- propensities because of an intermolecular electrostatic interaction created by the phosphomimetic substitution at the CTR. Our second aim will thus use FRET to verify the presence of this hypothesized intermolecular interaction and probe which residues contribute to it. Furthermore, fluorescence recovery after photobleaching (FRAP) will be employed to determine how these phosphomimetic substitutions alter the material properties of TDP-43 LCD drop...