Saposin-Lipoprotein Scaffolds for Structure Determination of Membrane Transporters

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Membrane proteins depend on their natural lipid environment for function, which makes them more difficult to study in isolation. A number of approaches that mimic the lipid bilayer of biological membranes have been described (nanodiscs, SMALPs), enabling novel ways to assay activity and elucidate structures of this important class of proteins. More recently, the use of saposin A, a protein that is involved in lipid transport, to form Salipro (saposin–lipid–protein) complexes was demonstrated for a range of membrane protein targets (Frauenfeld et al., 2016). The method is fast and requires few resources. The saposin–lipid–scaffold adapts to various sizes of transmembrane regions during self-assembly, forming a minimal lipid nanoparticle. This results in the formation of a well-defined membrane protein–lipid complex, which is desirable for structural characterization. Here, we describe a protocol to reconstitute the sarco-endoplasmic reticulum calcium ATPase (SERCA) into Salipro nanoparticles. The complex formation is analyzed using negative stain electron microscopy (EM), allowing to quickly determine an initial structure of the membrane protein and to evaluate sample conditions for structural studies using single-particle cryo-EM in a detergent-free environment.

OriginalsprogEngelsk
TitelMethods in Enzymology : A Structure-Function Toolbox for Membrane Transporter and Channels
RedaktørerChristine Ziegler
Antal sider15
Vol/bind594
ForlagAcademic Press
Udgivelsesår2017
Sider85-99
Kapitel3
ISBN (trykt)978-0-12-812353-9
DOI
StatusUdgivet - 2017
SerietitelMethods in Enzymology
Vol/bind594
ISSN0076-6879

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