Chapter 3: A fluorescent window into protein folding and aggregation in cells.

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Cellular environments impose evolutionary pressures on protein folding efficiency.
• Imperfect protein folding leads to aggregation, a hallmark of neurodegenerative diseases.
• Current methods for studying protein folding are often limited to in vitro conditions.

Purpose of the Study:

• To develop a method for directly monitoring protein folding and aggregation behavior within living prokaryotic and eukaryotic cells.
• To provide a quantitative approach for analyzing protein misfolding transitions and aggregate formation in vivo.

Main Methods:

• Incorporation of specific binding motifs for the FlAsH dye into the protein of interest.
• Utilizing distinct fluorescence signals generated by the dye binding to folded versus unfolded protein states.
• Applying the method to cellular retinoic acid-binding protein and a huntingtin fragment chimera in vivo.

Main Results:

• Demonstrated a strategy to label and monitor protein behavior directly within cells.
• Developed protocols for in vivo protein labeling and fluorescence signal detection.
• Achieved quantitatively interpretable data on protein folding transitions and aggregate formation.

Conclusions:

• The developed method enables direct, in vivo monitoring of protein folding and aggregation.
• This technique offers a valuable tool for studying the molecular mechanisms underlying neurodegenerative pathologies.
• The approach provides a quantitative assessment of protein misfolding and aggregate formation in cellular contexts.