Preserved transmembrane protein mobility in polymer-supported lipid bilayers derived from cell membranes.
Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, β-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms.
Department of Applied Physics, Chalmers University of Technology , SE-41296 Gothenburg, Sweden.
Hudson
H
Pace
Department of Applied Physics, Chalmers University of Technology , SE-41296 Gothenburg, Sweden.
Lisa
L
Simonsson Nyström
Discovery Sciences, AstraZeneca R&D Mölndal , SE-43183 Mölndal, Sweden.
Anders
A
Gunnarsson
Department of Applied Physics, Chalmers University of Technology , SE-41296 Gothenburg, Sweden.
Elizabeth
E
Eck
Department of Physical Science, Southern Utah University , Cedar City, Utah 84720 United States.
Christopher
C
Monson
Discovery Sciences, AstraZeneca R&D Mölndal , SE-43183 Mölndal, Sweden.
Stefan
S
Geschwindner
Discovery Sciences, AstraZeneca R&D Mölndal , SE-43183 Mölndal, Sweden.
Arjan
A
Snijder
Department of Applied Physics, Chalmers University of Technology , SE-41296 Gothenburg, Sweden.
Fredrik
F
Höök
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D005898:Glass / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009068:Movement; D010713:Phosphatidylcholines / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D013499:Surface Properties
D004129:Dimethylpolysiloxanes; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D011092:Polyethylene Glycols; C013830:baysilon; D016282:Aspartic Acid Endopeptidases; C028694:1-palmitoyl-2-oleoylphosphatidylcholine