Detergent-Free Technologies and Anatrace Lipids
Are Setting Higher Standards

The “classical” pipeline for membrane protein purification and structure determination typically involves the use of a detergent (commonly DDM) for the solubilization of membranes, purification using standard techniques, and crystallization in a detergent micelle using vapor diffusion or LCP methods. This crystallization step often involves multiple rounds of detergent exchange and crystallization screening to find a detergent which is suitable for nucleation and crystal growth(1).
 
With the increasing use of Cryo-EM for membrane protein structure determination in the past five years, this “classical” pipeline has changed slightly, with a number of detergent-free technologies being used for the structure determination step. These technologies include Amphipols, Nanodiscs, Polymers, and Saposins. In 2017, there were 73 unique membrane protein structures determined, 14 of which were determined using detergent-free systems. In 2018, detergent-free systems were used in 15 of the 110 unique membrane protein structures. Below, we’ll introduce each of these technologies, and provide some recent examples of their use in the literature. For a more comprehensive review, we highly recommend Jean-Luc Popot’s recent book “Membrane Proteins in Aqueous Solutions: From Detergents to Amphipols(2).

Amphipols: First described by Jean-Luc Popot in 1996, amphipols are a class of polymers that can stabilize membrane proteins in a detergent-free, aqueous solution(3). After the membrane protein is solubilized and purified using detergents, amphipols can replace the detergent, and excess detergent can be removed from the solution. The most well characterized amphipol is the anionic Amphipol A8-35, which has been used in over 25 Cryo-EM structures of membrane proteins including the recent structures of the Connexin-46 Gap Junction Channel(4), the XaxAB pore complex(5), and the TRPC4 TRP channel(6). Recently, the zwitterionic Amphipol PMAL-C8, developed in a collaboration between Charles Sanders and Anatrace, has been gaining traction with a number of recently published structures. These include the H. Pylori Urea Channel(7), the Mitochondrial Calcium Uniporter(8), and the TRPM7 TRP channel(9). Lastly, there is one published structure using a non-ionic amphipol (NAPol), the N. crassa TOM Core Complex(10).

Nanodiscs: Originally described in 2003 by Stephen Sligar, nanodiscs utilize a membrane scaffold protein (MSP) and exogenous lipids to allow for reconstitution of a membrane protein into a lipid environment(11). Similar to amphipols, the membrane protein first needs to be solubilized and purified using detergents prior to the reconstitution step. To date there have been over 10 unique Cryo-EM structures of membrane proteins determined using nanodiscs, including the OSCA1.2 ion channel(12), the Eukarytic OST complex(13), and the potassium channel, SthK(14).

Salipro: Described in 2016 by Jens Frauenfeld and Par Nordlind, saposin proteins form nanodisc-like structures in the presence of lipids, in which a membrane protein can be reconstituted into a detergent-free lipid environment(15). Unlike MSP nanodiscs, Salipro is a versatile tool that can adapt to proteins with different membrane cross-sections. There are two examples of Cryo-EM structures determined using Salipro to date, the mitochondrial calcium uniporter(16) and the TPC1 ion channel(17).

Polymers: In 2009, Tim Dafforn and Michael Overduin described the use of a styrene-maleic acid copolymer (SMA) to solubilize membrane proteins directly from the native lipid environment(18). Unlike other methods, detergents are not used at any point in the protein purification steps. There are two recent examples of SMAs being used for structure determination: the drug efflux pump AcrB(19)(20) and Alternative Complex III(21). Recently, the lab of Sandro Keller characterized a new polymer, diisobutylene/maleic acid (DIBMA), which can also solubilize and stabilize membrane proteins directly from the native lipid environment(22). DIBMA was released by Anatrace in September, 2018.

New Lipids from Anatrace

The most commonly used lipid mixture for nanodisc reconstitution is POPC : POPE : POPG in a 3:1:1 ratio which is typically used at a concentration of 0.1 mg / ml. To facilitate reconstitution into lipid nanodiscs, we are excited to offer our 3:1:1 POPC:POPE:POPG lipid mix. Simply solubilize the lipid mix in a detergent containing buffer and use in your experiments. Additionally, we are continuing to expand our offerings of synthetic phospholipids and are excited to announce the addition of DOPG, DMPE, POPS, and DPPS to our catalog.


 References:
  1. Vergis, JM et al. (2010) Anal Biochem 407(1), 1-11
  2. Popot, JL (2018) Membrane Proteins in Aqueous Solutions: From Detergents to Amphipols. Springer International Publishing. ISBN: 978-3-319-73146-9.
  3. Tribet, C., et al. (1996) Proc Natl Acad Sci U S A 93(26), 15047-15050.
  4. Myers, JB et al. (2018) Nature 564(7736), 372-377
  5. Schubert, E et al. (2018) Elife e38017, doi: 10.7554/eLife.38017
  6. Vinayagam, D et al. (2018) Elife e36615, doi: 10.7554/eLife.36615
  7. Cui, Y et al. (2019) Sci Adv 5(3), eaav8423
  8. Yoo, J et al. (2018) Science 361(6401), 506-511
  9. Duan, J et al. (2018) Proc Natl Acad Sci USA 115(35), E8201-E8210
  10. Bausewein, T et al. (2017) Cell 170(4), 693-700
  11. Bayburt, TH and Sligar, SG (2003) Protein Sci 12(11), 2476-2481
  12. Jojoa-Cruz, S et al. (2018) Elife e41845, doi: 10.7554/eLife.41845
  13. Wild, R, et al. (2018) Science 359(6375), 545-550
  14. Rheinberger, J et al. (2018) Elife e39775, doi: 10.7554/eLife.39775
  15. Frauenfeld, J et al. (2016) Nat Methods 13(4), 345-351
  16. Nguyen, NX et al. (2018) Nature 559(7715), 570-574
  17. Kintzer, AF et al. (2018) Proc Natl Acad Sci USA 115(39), E9095-E9104.
  18. Knowles, T. J. et al. (2009) J. Am Chem Soc. 131(22), 7484-7485.
  19. Parmar, M., et al. (2018) Biochim Biophys Acta. 1860(2), 378-383.
  20. Qiu, W, et al. (2018) Proc Natl Acad Sci USA 115(51), 12985-12990.
  21. Sun, C., et al. (2018) Nature 557(7703), 123-126.
  22. Oluwole, A. O. et al. (2017) Angew Chem Int Ed Engl. 56(7), 1919-1924.