PEG 1000 as a molecular switch: Concentration-dependent aggregation and refolding in a non-aggregation-prone β-barrel...

Area of Science:

• Biochemistry
• Protein Science
• Cellular Biology

Background:

• Macromolecular crowding profoundly impacts protein folding, stability, and aggregation in cellular settings.
• Cellular Retinoic Acid Binding Protein I (CRABP I) is a stable β-barrel protein with low aggregation tendency.

Purpose of the Study:

• To investigate the concentration-dependent effects of PEG 1000 on the temperature-assisted refolding of CRABP I.
• To analyze the structural, functional, and biological consequences of CRABP I refolding under varying PEG 1000 concentrations.

Main Methods:

• Spectroscopic and microscopic analyses to assess protein structure.
• Rheological, ligand-binding, and zeta potential measurements for functional and biophysical characterization.
• In vivo studies in Drosophila melanogaster to evaluate biological effects.

Main Results:

• Refolding without PEG yielded structurally heterogeneous intermediates.
• Low PEG 1000 concentrations induced amorphous aggregation, altered CRABP I structure, reduced retinoic acid binding, and increased cellular damage.
• Higher PEG 1000 concentrations prevented aggregation, stabilized CRABP I conformations, and preserved ligand-binding function.

Conclusions:

• PEG 1000 acts as a concentration-dependent molecular switch.
• It modulates the balance between protein aggregation and productive refolding of CRABP I.
• Understanding these effects is crucial for protein stability and cellular function studies.