Sunday, May 18, 2025
5/18/2025
Protein Structure, Dynamics, and Aggregation in Phase Separated Droplets - 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.
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