There are standards, and then there are higher
standards. and then there's NAPol

In last month’s newsletter, we highlighted several recent results using Biotinylated Amphipol A8-35, and launched our newest functionalized amphipol, Biotinylated PMAL-C8. To recap, amphipols are a class of polymers which can stabilize membrane proteins in a detergent-free, aqueous solution⁽¹⁾. To reconstitute a membrane protein in to amphipols, first the protein is solubilized and purified using detergents (e.g. DDM). Amphipols are then added to the solution and the detergents are removed, resulting in an amphipol reconstituted membrane protein.

With the “resolution revolution”⁽²⁾ in single particle electron cryo-microscopy (cryo-EM), there are more unique membrane protein structures being determined than ever before (for an overview, see our February, 2019 newsletter). Both Amphipol A8-35 and Amphipol PMAL-C8 have been instrumental in this effort, with over 25 unique membrane protein structures determined using these two molecules. Despite their utility, there are some limitations to these molecules. Amphipol A8-35 is anionic, and has been shown to have decreased solubility in acidic pH. The PMAL amphipols are zwitterionic, and the charge state varies based on pH (cationic at pH < 7, neutral at pH > 7). Both Amphipol A8-35 and the PMAL amphipols have reduced solubility in the presence of multivalent cations such as calcium⁽³⁾, and both are also incompatible with cell-free expression techniques for membrane proteins.

This month, we are continuing our highlight of amphipols by launching our newest addition to our amphipol family, Non-Ionic Amphipol (NAPol). First developed at the University of Avignon in the laboratory of Bernard Pucci⁽⁴⁾, and further characterized by the laboratory of Jean-Luc Popot at CNRS⁽⁵⁾, each repeating unit of the holotelomeric NAPol contains two glucose molecules and one undecyl chain. The non-ionic nature of this amphipol means that it is soluble across a wide pH range, and is compatible with multivalent cations. In these original studies, NAPol was used to reconstitute and stabilize the two model membrane proteins Bacteriorhodopsin and OmpX. Additionally, NAPol was shown to be compatible with cell-free synthesis of membrane proteins, as well as NMR studies.

Recently, the application of NAPol for the cryo-EM structure determination of membrane proteins was demonstrated. In August 2017, the lab Werner Kuhlbrandt at Max Plank Institute of Biophysics in Germany published the cryo-EM structure of the TOM Core Complex from Neurospora crassa⁽⁶⁾. In this study, the protein was solubilized and purified using DDM, and exchanged into NAPol for structure determination. Additionally, in January 2019, the lab of Jun Minagawa at National Institute of Basic Biology in Japan published the cryo-EM structure of the photosystem I supercomplex from Chlamydomonas reinhardtii⁽⁷⁾. Here, the protein was solubilized and purified in DDM, and exchanged into NAPol for structure determination. Interestingly, density of the NAPol can be seen in the Cryo-EM projection map. Most recently, the Minagawa Lab also published the structure of the large photosystem II-light harvesting complex II supercomplex of Chlamydomonas reinhardtii in August, 2019⁽⁸⁾. A NAPol-based purification method was able to yield the largest photosynthetic supercomplex to the highest percentage of intact configuration reported to date!

Check out the product page for the new Non-Ionic Amphipol (NAPol) here!

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There are standards, and then there are higherstandards. and then there's NAPol

There are standards, and then there are higher
standards. and then there's NAPol