Polyethylene Glycol-Mediated Crowding: Insights through Protein Dynamics and Phase Separation

Area of Science:

• Biophysics and Polymer Science
• Protein Dynamics and Macromolecular Crowding

Background:

• Polyethylene glycol (PEG) is a versatile model for protein-polymer interactions and a common macromolecular crowder.
• Different PEG molecular weights exhibit diverse biomolecular interactions, yet systematic comparisons as crowders are scarce.

Purpose of the Study:

• To comprehensively investigate the impact of various PEG molecular weights (PEG 1-20) on protein behavior and crowder characteristics.
• To elucidate the influence of PEG crowders on enzyme structure, dynamics, and phase separation phenomena.

Main Methods:

• Utilized circular dichroism, FRET, and solvation dynamics to study enzyme AK3L1 conformation and local dynamics.
• Employed fluorescence correlation spectroscopy and nanoparticle tracking analysis for diffusion measurements of proteins and crowders.
• Investigated PEG-induced phase separation using confocal microscopy and fluorescence recovery after photobleaching.

Main Results:

• Excluded-volume effects from PEGs induced AK3L1 structural compaction and increased local rigidity.
• PEG 10 demonstrated the strongest retardation of solvation dynamics, with crowder size significantly impacting phase separation.
• Dextran-PEG mixtures showed greater incompatibility and phase separation than Ficoll-PEG systems.

Conclusions:

• Detailed insights into how PEG crowders influence enzyme dynamics and protein-polymer interactions.
• Understanding the biophysical basis of crowder-induced condensate formation and solution properties of PEGs.