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Incorporation Of Membrane Proteins Into Lipid Bilayers For Scanning Transmission Electron Microscopy And Single Particle Reconstruction

Published online by Cambridge University Press:  02 July 2020

D.E. McAlduff ,
Y.M. Heng  and
F.P. Ottensmeyer
D.E. McAlduff
Affiliation:
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, ONM5G 2M9, Canada.
Y.M. Heng
Affiliation:
Electron Microscopy Facilities, MacMaster University Medical Center, Hamilton, ONL8N 3Z5, Canada.
F.P. Ottensmeyer
Affiliation:
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, ONM5G 2M9, Canada.
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Extract

The experimental approaches available to study the structure of most membrane proteins are limited. X-ray crystallography or electron crystallography rely on the availability or creation of 3D or 2D crystals to obtain high resolution structural information of membrane proteins. Single particleimaging of detergent solubilized protein is also possible, but removes the protein from its native environment. Torecreate suchan environment, we have developed a method which permits membrane proteins to be visualized in a lipid bilayer support.

A copper grid overlaid with a holey plastic film is vertically submerged into a lipid monolayer (Fig.l). This action creates planar lipid bilayers spanning 1 to 10 μm holes of the plastic film. These synthetic membranes have a thickness of approximately 40 Å, comparable to literature values for various cell membranes and are reasonably stable under a defocused beam in a transmission electron microscope (TEM)(Fig.2). Proteoliposomes with appropriate proteins in their membrane are added to the still-wet fresh lipid bilayers such that the liposomes fuse with the planar membrane (Fig.3).

Type
Biological Specimen Preparation
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 1314 - 1315
Copyright
Copyright © Microscopy Society of America

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References

References:

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6.The work was supported by funds from NSERC of Canada and MRC of Canada. The donation of P-gp by Dr. Frances Sharom of the University of Guelph is greatly appreciated.Google Scholar