Observing proteins as single molecules encapsulated in surface-tethered polymeric nanocontainers

Area of Science:

• Biochemistry and Biophysics
• Materials Science
• Polymer Chemistry

Background:

• Observing individual biomolecules is challenging due to diffusion.
• Immobilization techniques are crucial for single-molecule studies.
• Polymeric vesicles offer a potential solution for biomolecule encapsulation and observation.

Purpose of the Study:

• To assess the suitability of polymeric vesicles (polymersomes) for single-molecule protein folding studies.
• To investigate the stability and permeability of polymersomes under chemical stress.
• To demonstrate the application of polymersomes in monitoring protein unfolding and refolding dynamics.

Main Methods:

• Encapsulating individual proteins within amphiphilic triblock copolymer vesicles.
• Tethering vesicles to a cover slide surface for immobilization.
• Utilizing fluorescence spectroscopy with a photoinduced electron transfer (PET) sensitive dye (Atto655) to monitor protein conformational changes.
• Subjecting encapsulated proteins to chemical unfolding (GdnHCl) and refolding conditions.

Main Results:

• Polymeric vesicles demonstrated extreme stability, maintaining structural integrity under harsh unfolding conditions.
• Polymersomes were found to be permeable to guanidinium hydrochloride (GdnHCl), enabling chemical manipulation of encapsulated proteins.
• Individual protein unfolding and refolding transitions were successfully monitored in real-time using fluorescence changes.

Conclusions:

• Polymeric vesicles (polymersomes) are highly stable and permeable, making them excellent platforms for single-molecule biophysical studies.
• This approach enables detailed investigation of protein folding dynamics at the single-molecule level.
• The developed method provides a robust system for studying protein conformational changes under controlled conditions.