title: Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy.
abstract: Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet.
authors.forename: Ramakrishnan
authors.initials: R
authors.lastname: B Kumar
authors.affiliation: School of Technology and Health, KTH Royal Institute of Technology.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet; School of Technology and Health, KTH Royal Institute of Technology.
authors.forename: Hans
authors.initials: H
authors.lastname: Hebert
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet; School of Technology and Health, KTH Royal Institute of Technology; Caroline.Jegerschold@ki.se; cije@kth.se.
authors.forename: Caroline
authors.initials: C
authors.lastname: Jegerschöld
mesh_terms: D020285:Cryoelectron Microscopy; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D049329:Nanostructures / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins