Membrane proteins (GPCRs, ion channels, transporters, viral proteins, etc.) are stable in their lipid environment. Extraction from that environment for isolated structural or functional studies is a major obstacle. It requires destabilizing the lipid bilayer while at the same time solubilizing and stabilizing the membrane protein. Conventional denaturing methods cannot stabilize membrane domains as efficiently as the lipids found naturally in membranes and—much to the frustration of the user—affect the native state of the membrane protein. This often leads to loss of protein activity and prevents crystallization in an active conformation. An innovative detergent/surfactant-based approach has been developed that maintains the functional and structural integrity of membrane proteins. It does not require refolding, nor mutagenesis, truncation, or fusion, which can have significant impact on protein conformation. Instead of modifying the protein, this new approach modifies the chemical environment surrounding the protein. Membrane lipids are replaced by hydrophobic interactions with the novel detergent, and the protein is maintained in micelles as it transitions through hydrophilic interactions with solvents.

Calixarene-Based Extraction and Stabilization Detergents

A new class of anionic calixarene-based detergents have been designed to successfully stabilize extracted/purified membrane protein by maintaining a higher rigidity of the membrane domain and more closely mirroring the rigidity imposed by lipids in a bilayer membrane environment. Forming micelles of 5-24 nm, with critical micelle concentrations (CMCs) ranging from 0.05-1.5 mM, these detergents behave as mild surfactants. They structure membrane domains through multiple salt bridges with the positively charged residues found at the cytosol-membrane interface of membrane proteins (Figure 1). This network creates favorable hydrophobic interactions between detergent molecules and the membrane domain and strengthens membrane domain packing, which allows the membrane protein to remain in its native conformation in the absence of lipids. Additionally, pi stacking interactions between calixarene and aromatic residues create stability.

Figure 1. Proteins display a high degree of basic residues at the cytosol-membrane interface. Amphiphilic calixarene detergents (grey and black structures) accumulate here upon protein extraction, building a protective network of salt bridges with these basic residues in close proximity to the membrane region. Image sourced from Matar-Merheb et al. and used under CC BY 4.0.

The compounds are comprised of a rigid calix[4]arene backbone with three negatively charged methylene-carboxylate groups attached at the para position and a single hydrophobic tail of variable carbon chain length (Figure 2). This tail can range from 1-12 carbons and allows for the modification of hydrophobic properties needed for optimization during membrane protein extraction. Overall, this type of elongated tetrahedral structure favors the formation of spherical micelles. As weak acids, they can be used in a pH range of 5.5 to 14. Changes in pH can alter CMC, which can be taken advantage of for modulating micelle behavior.

Figure 2. Basic calixarene structure involves three aromatic rings substituted by methylene carboxyl groups at the para position and an aliphatic chain of variable length on the fourth phenolic group. Image sourced from Matar-Merheb et al. and used under CC BY 4.0.

These detergents may also bear protective glycoside or cholesterol-like groups. Classical detergents tend to strip away all lipids from the membrane protein, many of which are important for protein function. The use of lipid-bearing calixarene detergents circumvents this by providing a familiar environment that permits membrane protein stability and functionality. Cayman offers the following calixarene surfactants for the purification of active membrane proteins.

Calixarene surfactants can be used throughout the purification process, including metal affinity and gel filtration chromatographies. Once solubilized to a protein/lipid/detergent complex, proteins can then be purified in detergent (successfully exchanged with a more conventional detergent, if required) and reconstituted into proteoliposomes, nanodisc/peptidisc complexes, or amphipol/protein complexes.

Therapeutic Targets

Membrane proteins are directly involved in a wide variety of cellular processes and represent a majority of therapeutic targets. Unlocking their structure/function details is an essential step towards providing the most accurate information needed throughout drug discovery. Indeed, correct membrane protein conformations are required for structure-based drug design, ligand screening, antibody discovery, etc. These new detergents have already been used to extract several membrane proteins from different origins that are notoriously sensitive to detergent extraction and have been shown to retain protein functionality (Figure 3). Cayman provides several functional purified membrane proteins extracted using this technique.

Purified Membrane Proteins

Figure 3. Simulation of multidrug efflux pump subunit AcrB (E. coli strain K12) (PDB 5YIL) and adenosine A_2A receptor (PDB 5WF5) being extracted from a lipid membrane bilayer and held in active conformations using calixarene-based detergents.

View complete details on all protein purification tools available from Cayman

Publications

1. Matar-Merheb, R., Rhimi, M., Leydier, A., et al. Structuring detergents for extracting and stabilizing functional membrane proteins. PLoS One. 6(3), e18036 (2011).

2. Rosati, A., Basile, A., D'Auria, R., et al. BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages. Nat. Commun. 6(8695) (2015).

3. Desuzinges Mandon, E., Agez, M., Pellegrin, R., et al. Novel systematic detergent screening method for membrane proteins solubilization. Anal. Biochem. 517, 40-49 (2017).

4. Agez, M., Schultz, P., Medina, I., et al. Molecular architecture of potassium chloride co-transporter KCC2. Scientific Reports 7, 16452 (2017).

5. Hardy D., Desuzinges Mandon, E., Rothnie, A., et al. The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein. Methods S1046-2023(17), 30434-30436 (2018).

6. Igonet, S., Raingeval, C., Cecon, E., et al. Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor. Scientific Reports8, 8142 (2018).

7. Boussambe, GNM., Guillet, P., et al. Fluorinated diglucose detergents for membrane-protein extraction. Methods. 147, 84-94 (2018). (PMID: 29857192)

Patents

1. Coleman, A.W., Mbemba, C., Falson, P.G., et al. Method for selectively extracting membrane proteins using calixarenes, WO2009144419.

2. Falson, P.G., Coleman, A.W., Matar, M.R., et al. Additive for the crystallization of proteins, use and process, WO2010116055.

3. Dauvergne, J. Synthesis of amphiphilic calixarene glycoside detergents and use of the same for extracting and stabilizing native functional membrane proteins, WO2015158575.

4. Dauvergne, J. Industrial process for the synthesis of calixarene compounds, WO2016020603.

5. Rosa-Calatrava, M., Traversier. A. Desuzinges-Mandon, É. Method for preparing vaccine antigen, resulting vaccine and uses, WO2016030635.

6. Dauvergne, J. and Jawhari, A. Steroidic calixarene compounds, their synthesis and use for extraction, solubilization and/or stabilization of native and functional membrane proteins, WO2017134087.