title: Lipid nanotechnologies for structural studies of membrane-associated proteins.
abstract: We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment.
authors: Department of Neuroscience and Cell Biology, The University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555; Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555.
authors: Svetla
authors: S
authors: Stoilova-McPhie
authors: Kirill
authors: K
authors: Grushin
authors: Daniela
authors: D
authors: Dalm
authors: Jaimy
authors: J
authors: Miller
mesh_terms: D002462:Cell Membrane / Q000737:chemistry; D020285:Cryoelectron Microscopy / Q000379:methods*; D005169:Factor VIII / Q000737:chemistry*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D043942:Nanotubes; D010718:Phosphatidylserines / Q000737:chemistry; D011487:Protein Conformation
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D010718:Phosphatidylserines; D005169:Factor VIII
keywords: cryoelectron microscopy; lipid nanodisks; lipid nanotubes; membrane-associated proteins; protein structure