# Solving the Phase Problem of TDP-43 and ALS-Associated Variants

> **NIH NIH F31** · UNIVERSITY OF PENNSYLVANIA · 2021 · $33,253

## Abstract

One of biology's central conundrums is how the cell regulates complex biochemical reactions in time and space.
Cells have solved this problem by producing compartments, namely organelles, comprised of distinct chemical
environments. Emerging evidence suggests that multiple "membrane-less" organelles within the cell assemble
via phase separation and behave like droplets within an aqueous environment. In vivo and in vitro, such phase
separation is primarily driven by intrinsically disordered domains or proteins (IDPs). The structural plasticity of
IDPs allows them to adopt various structural conformations, generating multiple, weakly adhesive, inter- and
intramolecular interactions. Unfortunately, this conformation flexibility comes at the cost of an increased risk of
protein jamming or aggregation. Evidence suggests that imbalances between the thermodynamic drive to
undergo phase separation and the established opposing aggregation control machinery could lead to disease.
For example, amyotrophic lateral sclerosis (ALS) is believed to arise from aberrant phase separation of
transactive response (TAR) element DNA binding protein of 43 kDa (TDP-43). TDP-43 is an essential RNA
binding protein that plays a fundamental role in mRNA splicing, transport, and stability. Despite having a vast
knowledge of the individual domains of TDP-43, there has been relatively little study of the full-length protein that
is found in biologically relevant phase separation. In particular, the structural and sequence-specific basis
of TDP-43 phase separation is not well understood and remains elusive. Our lab has a very successful
track-record with incorporating hydrogen/deuterium exchange mass spectrometry (HXMS) to solve emerging
biological questions. The overall objective of this proposal is to utilize HXMS to understand the structural and
molecular cues that trigger TDP-43 phase separation. I hypothesize that there are small, 3-5 amino acid long,
regions within the N-terminal, first RNA-recognition motif, and C-terminal domains of TDP-43 that are required
for phase separation. Additionally, this proposal will look at linking the structural dynamics of ALS-disease
variants with their increased propensity to phase separate. I hypothesize that certain mutations perturb the
structure of the C-terminal domain of TDP-43, producing larger or additional contact points for self-association,
leading to an increased degree of phase separation. My proposed experiments will provide a basic
understanding of how full-length and variant TDP-43 behaves in solution and lay the foundation for the structure-
based development of efficient inhibitors.

## Key facts

- **NIH application ID:** 10252768
- **Project number:** 5F31GM134591-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Nikaela Whitney Bryan
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $33,253
- **Award type:** 5
- **Project period:** 2020-08-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10252768

## Citation

> US National Institutes of Health, RePORTER application 10252768, Solving the Phase Problem of TDP-43 and ALS-Associated Variants (5F31GM134591-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10252768. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*