Biophysical characterization of membrane proteins in nanodiscs

详细信息    查看全文

• 作者：Sayaka Inagaki^a ; ^{inagakisn@ninds.nih.gov} ; Rodolfo Ghirlando^b ; ^{rodolfo.ghirlando@nih.gov} ; Reinhard Grisshammer^a ; ^{rkgriss@helix.nih.gov}
• 关键词：2xYT ; double strength YT medium ; AUC ; analytical ultracentrifugation ; CHAPS ; 3-[(3-cholamidopyropyl)dimethylammonio]-1-propanesulfonate ; CHS ; cholesteryl hemisuccinate Tris salt ; DDM ; n-dodecyl-¦Â-d-maltoside ; DLS ; dynamic light scattering ; E. coli ; Esche
• 刊名：Methods
• 出版年：2013
• 出版时间：1 March, 2013
• 年：2013
• 卷：59
• 期：3
• 页码：287-300
• 全文大小：897 K

文摘

Nanodiscs are self-assembled discoidal phospholipid bilayers surrounded and stabilized by membrane scaffold proteins (MSPs), that have become a powerful and promising tool for the study of membrane proteins. Even though their reconstitution is highly regulated by the type of MSP and phospholipid input, a biophysical characterization leading to the determination of the stoichiometry of MSP, lipid and membrane protein is essential. This is important for biological studies, as the oligomeric state of membrane proteins often correlates with their functional activity. Typically combinations of several methods are applied using, for example, modified samples that incorporate fluorescent labels, along with procedures that result in nanodisc disassembly and lipid dissolution. To obtain a comprehensive understanding of the native properties of nanodiscs, modification-free analysis methods are required. In this work we provide a strategy, using a combination of dynamic light scattering and analytical ultracentrifugation, for the biophysical characterization of unmodified nanodiscs. In this manner we characterize the nanodisc preparation in terms of its overall polydispersity and characterize the hydrodynamically resolved nanodisc of interest in terms of its sedimentation coefficient, Stokes¡¯ radius and overall protein and lipid stoichiometry. Functional and biological applications are also discussed for the study of the membrane protein embedded in nanodiscs under defined experimental conditions.