Preserved Transmembrane Protein Mobility in Polymer-Supported Lipid Bilayers Derived from Cell Membranes
Artikel i vetenskaplig tidskrift, 2015

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.

Författare

Hudson Pace

Chalmers, Teknisk fysik, Biologisk fysik

Lisa Simonsson Nyström

Chalmers, Teknisk fysik, Biologisk fysik

Anders Gunnarsson

AstraZeneca AB

Elizabeth Eck

Chalmers, Teknisk fysik, Biologisk fysik

C. Monson

Southern Utah University

S. Geschwindner

AstraZeneca AB

Arjan Snijder

AstraZeneca AB

Fredrik Höök

Chalmers, Teknisk fysik, Biologisk fysik

Analytical Chemistry

0003-2700 (ISSN) 1520-6882 (eISSN)

Vol. 87 18 9194-9203

Ämneskategorier

Cellbiologi

Biofysik

DOI

10.1021/acs.analchem.5b01449

PubMed

26268463

Mer information

Senast uppdaterat

2018-11-30