The aim of the course is to provide students with practical knowledge necessary for extraction, purification and characterization of membrane proteins in the form of styrene-maleic acid (SMA) bounded nanodiscs. Techniques to purify and characterise the resulting protein encapsulated nanodiscs will be briefly covered.
In this flash course we will illustrate the method for KcsA from E.coli. Bringing your own samples is possible. Please contact us first if you want to do this.
Membrane solubilisation typically requires the use of detergents. This is not an ideal situation as the native lipid environment is stripped away by detergents, which may lead to protein misfolding or aggregation. A promising alternative is the use of SMA copolymers . These polymers are capable of solubilising membrane proteins into nanodiscs together with surrounding lipids. Proteins in nanodiscs can thus be purified and characterized in their native lipid environment. In addition it is possible to study protein-lipid, protein-protein, and protein-ligand interactions.
SMA is an amphiphilic copolymer consisting of styrene and maleic acid subunits. The ratio of each of these can be varied (i.e., 1:1, 2:1, 3:1..) as well as the total length of the polymer. The polymers are initially synthesized as maleic anhydride (SMAnh), which can then be hydrolysed to form the active maleic acid groups. The type of SMA used is important for the efficiency of solubilisation: commonly utilized is the SMA 2:1 variant, with a weight average Mw ~10kD.
1 day in total (includes overnight incubation)
Adrian Kopf (email@example.com)
* Dörr, J. M., Scheidelaar, S., Koorengevel, M. C., Dominguez, J. J., Schäfer, M., van Walree, C. A., & Killian, J. A. (2016). The styrene–maleic acid copolymer: a versatile tool in membrane research. European Biophysics Journal, 45, 3–21. http://doi.org/10.1007/s00249-015-1093-y.