title: Taming membranes: functional immobilization of biological membranes in hydrogels.
abstract: Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, protein translocation activity by the membrane embedded protein conducting channel of bacteria, SecYEG, in association with the soluble motor protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.
authors.affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, AG Groningen, The Netherlands.
authors.forename: Ilja
authors.initials: I
authors.lastname: Kusters
authors.forename: Nobina
authors.initials: N
authors.lastname: Mukherjee
authors.forename: Menno R
authors.initials: MR
authors.lastname: de Jong
authors.forename: Sander
authors.initials: S
authors.lastname: Tans
authors.forename: Armağan
authors.initials: A
authors.lastname: Koçer
authors.forename: Arnold J M
authors.initials: AJ
authors.lastname: Driessen
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D020100:Hydrogels / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D053835:Unilamellar Liposomes / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D020100:Hydrogels; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D053835:Unilamellar Liposomes; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins