Observing proteins as single molecules encapsulated in surface-tethered polymeric nanocontainers.
Immobilizing biomolecules provides the advantage of observing them individually for extended time periods, which is impossible to accomplish for freely diffusing molecules in solution. In order to immobilize individual protein molecules, we encapsulated them in polymeric vesicles made of amphiphilic triblock copolymers and tethered the vesicles to a cover slide surface. A major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein-folding studies. The fact that polymeric vesicles possess an extreme stability under various chemical conditions is supported by our observation that harsh unfolding conditions do not perturb the structural integrity of the vesicles. Moreover, polymerosomes prove to be permeable to GdnHCl and, thereby, ideally suited for unfolding and refolding studies with encapsulated proteins. We demonstrate this with encapsulated phosphoglycerate kinase, which was fluorescently labeled 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
Tobias
T
Forschungszentrum Jülich, Molecular Biophysics, Jülich, Germany.
Katranidis
Alexandros
A
Atta
Diaa
D
Gregor
Ingo
I
Enderlein
Jörg
J
Grzelakowski
Mariusz
M
Rigler
Per
P
Meier
Wolfgang
W
Fitter
Jörg
J
D005456:Fluorescent Dyes / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D055668:Photochemical Processes; D011108:Polymers / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D012441:Saccharomyces cerevisiae; D013499:Surface Properties
D005456:Fluorescent Dyes; D005656:Fungal Proteins; D055767:Immobilized Proteins; D008081:Liposomes; D011108:Polymers