# Protein Structure, Dynamics, and Aggregation in Phase Separated Droplets

> **NIH NIH R35** · UNIVERSITY OF NOTRE DAME · 2024 · $1

## Abstract

Protein Structure, Dynamics, and Aggregation in Phase Separated Droplets
Abstract
Amyotrophic lateral sclerosis (ALS) is a severe and deadly disease. In recent years, it has been discovered that
a key mechanism of disease progression lies in liquid-liquid phase separation (LLPS) of a number of peptides
and proteins. Indirect evidence has also emerged that LLPS can induce protein folding/aggregation into amyloid-
like hydrogels in a number of different diseases, including ALS, type-2 diabetes, and Alzheimer’s disease. We
recently reported direct in-situ evidence that phase separation induces a folding transition for peptide and
proteins derived from ALS. This proposal aims to build on that work to develop and apply spectroscopic tools for
in-situ characterization of protein structure, dynamics, and solvation within phase-separated droplets, in order to
identify the structure and mechanism of formation of these folded proteins and gels, and to study how these
changes relate to the disease state of ALS. To accomplish this, we will use two-dimensional infrared
spectroscopy (2DIR), infrared microscopy, and 2DIR microscopy, to probe changes in secondary structure and
hydration of peptides and proteins within droplets, and understand the fundamental biophysical processes
involved in protein LLPS. Key questions that we aim to answer are: What role does solvation serve in the driving
forces governing LLPS? Can volumetric crowding in polymer dense LLPS droplets promote changes in protein
secondary structure? Can LLPS drive protein folding/aggregation into potentially toxic amyloid states? We will
be able to answer these questions for in-situ studies, something currently not possible with other techniques.
The strategy outlined in this proposal is designed with the long-term goal of building a research program that
can perform structural studies in complex biophysical systems, turning the full suite of structure sensitive
observables in nonlinear IR spectroscopy towards addressing questions in whole cell systems.

## Key facts

- **NIH application ID:** 10915645
- **Project number:** 5R35GM150868-02
- **Recipient organization:** UNIVERSITY OF NOTRE DAME
- **Principal Investigator:** Arnaldo L Serrano
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1
- **Award type:** 5
- **Project period:** 2023-09-01 → 2025-02-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10915645

## Citation

> US National Institutes of Health, RePORTER application 10915645, Protein Structure, Dynamics, and Aggregation in Phase Separated Droplets (5R35GM150868-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10915645. Licensed CC0.

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