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Polymerosomes and Protein Encapsulation

Polymerosomes

  • synthetic ABA triblock copolymer

  • vesicle size: Ø 100 - 200 nm

  • remarkably stable nanocontainer with a long life time

  • straight forward entrapment of single molecules under physiological conditions

  • during production of polymerosomes

  • no interaction of encapsulated proteins with the vesicle surface

  • vesicles permeable for guanidine hydrochloride (protein unfolding inside the vesicle)

  • immobilisation to a PEG covered surface employing a streptavidin biotin binding assay

polymerosome_bild_1


Scheme of a polymer vesicle immobilized via biotin and streptavivin on a PEG functionalized cover side

Protein encapsulation

Entrapment of protein molecules in lipid vesicles or polymerosomes solves two crucial issues/challenges in single molecule spectroscopy

  • immobilisation and prolonged observation times without influencing the molecule of interest due to direct attachment

  • high local substrate/ligand concentration inside the compartment necessary due to micromolar substrate/ligand affinities and low overall concentration required for low fluorescent background intensity allowing single molecule detection sensitivity

Polymerosomes also hold a chaperone like property in folding experiments allowing to monitor unfolding/refolding transitions of encapsulated multi-domain proteins in the absence of aggregation which otherwise do not refold.

We demonstrated the advantages of using polymerosomes in unfolding/refolding studies for immobilization and encapsulation of single proteins by encapsuling phosphoglycerate kinase (PGK). PGK was unfolded and refolded by alternating concentrations of guanidine hydrochloride. The protein was fluorescently labeled specifically with Atto655, a dye that exhibits pronounced photoinduced electron transfer (PET) to a nearby tryptophan residue in the native state. Under unfolding conditions, PET was reduced, and we monitored alternating unfolding and refolding conditions for individual encapsulated proteins.


Rosenkranz et al., ChemBioChem 2009, 10, 702-709


Contact: Dr. Iris v. d. Hocht



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