Visualizing the Multistep Process of Protein Aggregation in Live Cells.

Area of Science:

• Biochemistry and Molecular Biology
• Chemical Biology
• Cellular Biology

Background:

• Protein aggregation involves misfolded proteins forming insoluble aggregates, with intermediate species like misfolded protein oligomers being poorly understood.
• Existing methods are insufficient for detecting and studying these transient oligomeric species within live cells.

Purpose of the Study:

• To develop novel chemical methods for characterizing misfolded protein oligomers in living cells.
• To enable the direct monitoring and differentiation of various protein aggregation states.

Main Methods:

• Development of the AgHalo sensor using destabilized HaloTag variants and solvatochromic fluorophores to detect misfolded oligomers.
• Extension to the AggTag method, fusing proteins of interest with self-labeling tags (HaloTag, SNAP-tag) and employing molecular rotor-based fluorophores.
• Design of novel triode-like fluorophores using physical and computational chemistry to distinguish between soluble oligomers and insoluble aggregates based on fluorescence emission.

Main Results:

• Successfully developed fluorogenic probes (AgHalo and AggTag) for detecting misfolded protein oligomers in live cells.
• Established methods to differentiate between misfolded oligomers and insoluble aggregates using distinct fluorophore classes.
• Demonstrated the ability to monitor protein aggregation pathways and intermediate species in real-time within cellular environments.

Conclusions:

• The developed chemical methods provide unprecedented tools for studying misfolded protein oligomers and their role in protein aggregation.
• These advancements facilitate research into currently unanswered questions regarding protein misfolding and aggregation dynamics.
• Future work will focus on quantitative analysis and extending these methods to study protein and RNA condensates.