Protein folding stability and dynamics imaged in a living cell

Area of Science:

• Biophysics
• Cell Biology
• Biochemistry

Background:

• Biomolecular dynamics and stability are typically studied in vitro, with findings extrapolated to cellular function.
• Understanding in vivo biomolecular behavior is crucial for explaining cellular processes.

Purpose of the Study:

• To introduce Fast Relaxation Imaging (FreI), a novel technique for investigating biomolecular dynamics and stability within living cells.
• To demonstrate FreI's capability in measuring protein folding kinetics and thermodynamics in real-time within a cellular context.

Main Methods:

• FreI combines fluorescence microscopy with rapid temperature jumps to achieve high spatiotemporal resolution.
• The method was applied to a FRET probe-labeled phosphoglycerate kinase construct in two human cell lines.
• Measurements were performed across a temperature range of 23-49°C.

Main Results:

• FreI successfully measured reversible fast folding kinetics and folding thermodynamics of the protein construct within living cells.
• The cellular environment was found to significantly influence protein stability and folding rates compared to in vitro conditions.
• The study provides quantitative data on protein folding dynamics in vivo.

Conclusions:

• FreI offers a powerful tool for studying biomolecular dynamics and stability directly within living cells.
• The cell's microenvironment plays a critical role in modulating protein folding and stability.
• FreI has broad applicability for studying protein-protein interactions, heat-shock responses, and comparative analyses across cell populations or organisms.