AggFluor: Chemical Modulation of Fluorescent Protein Chromophores Enables Visualization of the Multi-Step Protein Aggregation Process in Live Cells
Open Access
- Author:
- Wolstenholme, Charles
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 21, 2021
- Committee Members:
- Xin Zhang, Chair & Dissertation Advisor
Ying Gu, Outside Unit & Field Member
Scott Showalter, Major Field Member
Tae-Hee Lee, Major Field Member
Philip Bevilacqua, Program Head/Chair - Keywords:
- Fluorophores
GFP
Biochemistry
Organic Chemistry
Protein Misfolding
Neurodegenerative Disease - Abstract:
- In this dissertation, I plan to introduce four main aims: (1) Relevant background information on misfolding, aggregation and phase-separation in protein misfolding diseases. Along with traditional fluorescent and microscopic techniques to visualize a protein-of-interest aggregation are used, and how my work reveals more critical details regarding this multistep aggregation pathway. (2) My work to help develop a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photophysical analysis, X-ray crystallography, and theoretical calculation, to show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment protein aggregates. Biorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allowed for fluorogenic detection of both misfolded oligomeric and aggregated species in live cells. (3) The development of a chemical toolbox, AggFluor, that enables live cell imaging and differentiation of complex aggregated conformation states. Based on the chromophore of green fluorescent protein, AggFluor is made up of a series of rationally designed molecular-rotor fluorophores that span a broad spectrum of viscosity sensitivity. This feature allowed us to develop a dual-color imaging strategy to distinguish unfolded protein oligomers from insoluble aggregates in live cells. (4) The advancement of an AgGlow method, which is composed of a series of small molecule fluorophores that are useful in applications including detecting and quantifying pre-fibril protein oligomers and insoluble protein aggregates in high throughput technologies and in intact live cells. And the design of a group of synthetic probes that detect the formation of membrane-less organelles by showing a significant increase in fluorescent intensity when incorporated into phase-separated droplets relative to a homogenous control.