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Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
Published on: April 25, 2021
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Quantitative interrogation of protein co-aggregation using multi-color fluorogenic protein aggregation sensors.
Yulong Bai1,2, Wang Wan1, Yanan Huang1
1CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences 457 Zhongshan Road Dalian 116023 China liuyu@dicp.ac.cn wanwang@dicp.ac.cn.
Chemical Science
|July 5, 2021
Summary
This study introduces a novel dual-color assay to track protein co-aggregation in neurodegenerative diseases. The assay reveals how mutant proteins destabilize wild-type counterparts, offering insights into disease mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Protein co-aggregation is a hallmark of neurodegenerative diseases, yet understanding these complex interactions is difficult.
- Deconvoluting the biochemical processes underlying simultaneous protein aggregation remains a significant challenge in disease research.
Purpose of the Study:
- To develop a novel dual-color fluorogenic thermal shift assay for simultaneous monitoring of two protein aggregation events.
- To quantitatively assess the thermodynamic stability of proteins during co-aggregation.
- To investigate the influence of small molecule ligands on protein co-aggregation dynamics.
Main Methods:
- Development of multi-color fluorogenic protein aggregation sensors with minimal fluorescence crosstalk.
- Utilizing sortase-tag technology for orthogonal conjugation of sensors to distinct proteins.
- Quantification of melting temperature shifts in a heterozygous model system to determine thermodynamic stability changes.
- In vitro and live-cell imaging experiments to visualize protein co-aggregation interplay.
Main Results:
- The assay successfully enabled simultaneous reporting and quantitative analysis of two protein aggregation events.
- Mutant proteins were found to significantly compromise the thermodynamic stability of wild-type proteins, but not vice versa.
- Small molecule ligands demonstrated selective and differential interference with protein co-aggregation interactions.
- The developed sensors were validated for visualizing inter-protein influence during co-aggregation in live cells.
Conclusions:
- The dual-color fluorogenic thermal shift assay provides a powerful tool for studying complex protein co-aggregation.
- This method elucidates the differential impact of mutant proteins on wild-type stability, offering new perspectives on neurodegenerative disease pathogenesis.
- The findings pave the way for developing targeted therapeutics by understanding ligand interactions in protein aggregation.
