Lipid nanotechnologies for structural studies of membrane-associated proteins.
Proteins
; 82(11): 2902-9, 2014 Nov.
Article
em En
| MEDLINE
| ID: mdl-24957666
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 â¼20 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 â¼10 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.
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MEDLINE
Assunto principal:
Fator VIII
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Microscopia Crioeletrônica
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Nanotecnologia
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Proteínas de Membrana
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article