Background

Membrane proteins (MPs) are important components of the cell membrane, which play an important role in a variety of cellular functions, such as material transport, signal transduction and intercellular recognition. The membrane proteins encoded in the human genome account for about 25% of the total proteome. These membrane proteins are associated with a variety of diseases such as cystic fibrosis, atherosclerosis, Parkinson's disease and Alzheimer's disease. Membrane proteins exposed to extracellular areas are potential targets for many drugs. Membrane proteins are estimated to be the targets of 60% drugs, such as ABC transporters, nucleoside transporters, G protein-coupled receptors, caveolin-1, etc. Some membrane proteins regulate the cellular response so that the drug enters the cell directly, or helps the drug enter the cell in the form of a channel to exert its effect, while others actively pump the drug out of the cell. Therefore, membrane proteins can greatly affect the therapeutic effect of drugs.

As the most promising potential target group of drugs, the separation and purification of membrane proteins is of great significance for the study of its structure and function and the development of new drugs. It has been found that membrane proteins need to maintain the lipid bilayer or lipoid environment in order to maintain their natural conformation, but this environment is difficult to simulate in vitro, and membrane proteins have obvious tendency of aggregation and denaturation in aqueous solution. Therefore, the structural biology of membrane proteins lags behind. At present, only 2% to 3% of the proteins with known structures are membrane proteins. In addition, the expression of membrane proteins is generally low, so it is difficult to separate and purify enough target membrane proteins. How to separate and purify membrane proteins and obtain stable structure is a difficult problem in the study of membrane proteins. In view of the phenomenon that membrane proteins are easy to aggregate and denature after they are separated from the natural membrane environment, researchers proposed a detergent strategy based on surfactants to separate membrane proteins by using its micelles, but there are some problems in the process of application. Furthermore, amphiphilic systems such as liposomes and nanodiscs were proposed to simulate the natural membrane environment, which ensured the conformational and functional integrity of membrane proteins in the process of separation and purification to a certain extent. In recent years, polymer stabilization technology has emerged to protect the local lipid environment around membrane proteins.

Detergent Stabilization Strategy

The early stabilization of membrane protein was mainly realized by detergent strategy. Detergent is a general term for a class of mixtures dominated by surfactants, which can reduce the interfacial tension between two immiscible liquids. The whole molecular structure of detergent consists of hydrophilic polar head group and hydrophobic non-polar tail group. In aqueous solution, the cell membrane exists in the form of lipid bilayer, and the detergent molecule exists as micelle when it is higher than the critical micelle concentration (CMC). When the cell membrane and detergent micelles are mixed, the detergent molecules are first bound to the cell membrane surface in a non-cooperative manner. As the detergent concentration increases, the detergent molecules are incorporated from the cell membrane surface into the interior of the lipid bilayer. At this time, the non-cooperative binding changes to cooperative binding, and the cooperative and non-cooperative binding of detergent to the cell membrane is called the cross-bilayer mechanism. As more detergent is incorporated into the membrane, lipid-detergent mixed micelles form. Then the cell membrane is lysed, and the detergent successfully extracts the membrane proteins to form micelles.

Fig. 1 Different membrane protein stabilization strategies using artificial membranes. (Sgro GG, 2018)

Traditional detergents include N-octyl-β-D-glucopyranoside (OG), N-decyl-β-D-maltoside (DM), N-dodecyl-β-D-maltoside (DDM), N-nonyl-β-D-glucopyranoside (NG) and lauryl dimethylamine oxide (LDAO). DDM, as the most classical detergent, is usually the preferred detergent for membrane protein extraction. Different detergents have different tendencies for the separation and purification of membrane proteins. DDM has a good effect on the separation of transporters and respiratory complexes, while OG has a good effect on the separation of channel proteins, and LDAO will lead to the accumulation of most transporters. In LDAO, the voltage-dependent anion channel protein can maintain a stable structure. In addition, different types of detergents require different concentrations of phosphate to extract membrane proteins. At higher phosphate concentration, zwitterionic detergent LDAO has higher extraction efficiency than nonionic detergent DDM. Different types of detergents have different stability to proteins, and it is estimated that only 20% of the membrane proteins can remain stable in LDAO. In contrast, most of the proteins in nonionic detergents DDM are more stable than LDAO, but the extraction efficiency of nonionic detergents is often lower than that of zwitterionic detergents. In the application of traditional detergents, the common problem described by researchers is that the stability of membrane proteins in micelles is not high, it is difficult to maintain the natural conformation of membrane proteins, which limits the study of the structure and function of membrane proteins and their downstream applications.

In order to solve the problem of low stability of membrane proteins in traditional detergents, researchers have developed a series of novel detergents. Compared with traditional detergents, they can stabilize membrane proteins better. Although the novel detergents improve the stability of membrane proteins in the process of separation and purification to some extent, both novel and traditional detergents have some common defects.

• Detergent selection for the isolation, purification, and stabilization of unknown membrane proteins requires extensive screening.
• When membrane proteins are separated and purified by detergents in the form of micelles, membrane proteins often lose their natural interaction with lipids and other proteins.
• The detergents with good effect of stable separation of membrane proteins sometimes interfere with downstream applications. Therefore, it is impossible to predict which detergent is most effective for a given protein, which needs to be determined by repeated experiments.

Detergent Products

Our high-purity detergent products:

Artificial Membrane Stabilization Strategy

In view of the shortcomings of the detergent strategy, the researchers proposed a new artificial membrane strategy of amphiphilic system to simulate the natural membrane environment. Inspired by the emerging nanoscience, this strategy fully takes into account the complexity of phospholipid bilayers and the importance of maintaining the activity of membrane proteins to ensure stable separation and purification of membrane proteins in the local membrane environment. The basic principle of artificial membrane stabilization of membrane proteins is to prepare the phospholipid components contained in the cell membrane into various forms such as liposomes, bicelles and nanodiscs using different reaction conditions, and mix them with purified or recombinant membrane proteins in a certain proportion. This can realize the reconstruction of the membrane protein-artificial membrane mosaic model.

Liposomes

Liposomes disperse amphiphilic molecules such as phospholipids and sphingolipids in the aqueous phase. The hydrophobic tails of the molecules tend to cluster together and avoid the aqueous phase. The hydrophilic head is exposed to the aqueous phase, forming a closed vesicle with a bilayer structure. Liposomes can be prepared from pure lipids or mixed lipids, and the proportion can be adjusted according to the target protein. The size of the liposome can also be adjusted by the dispersion preparation step. Liposomes have unique advantages for transporters such as nucleoside transporters. Recombinant liposomes can be used to test the functional integrity of membrane proteins by selectively concentrating lipid substrates. Similarly, if the protein liposome is formed in the presence of the substrate, it can also be evaluated in reverse. In addition, the proportion and type of lipids in liposomes will greatly affect the activity, stability and crystallization ability of membrane proteins. Liposomes can promote crystallization by stabilizing connections between protein folds, monomers, and subunits, as well as through liposome-mediated lattice contacts to determine the structure and function of membrane proteins and study corresponding drug targets.

Bicelles

Bicelles are self-assembled disc structure formed by a mixture of long-chain phospholipids and short-chain lipids (or detergents). Long-chain phospholipids form bilayers containing membrane proteins, which are then stabilized by short-chain lipids located at the edge of the bilayer. At present, the most commonly used is the combination of dihexyl phosphatidylcholine (DHPC, short-chain) and dicarbonyl phosphatidylcholine (DMPC, long-chain). Bicelles have different conformations according to the ratio of long-chain phospholipids and short-chain lipids, temperature, pH value and salt concentration. Bicelles are intermediate forms between liposome vesicles and classical detergent micelles, which combines the advantages of these two systems. Compared to liposomes, bicelles are easier to achieve uniform mixing. Compared with classic detergent micelles, the amount of detergent in bicelles is lower, and bicelles are closer to the natural membrane environment. However, bicelles also have limitations, such as the appropriate proportion of long-chain phospholipids and short-chain lipids is often difficult to control. The detergent exchange step in bicelles sometimes leads to the accumulation of membrane proteins, which is limited by the problems related to the use of detergents.

Nanodiscs

Nanodiscs consists of phospholipids and amphiphilic helical proteins, also known as membrane scaffold proteins (MSP). Generally, a bilayer containing membrane proteins is formed by 130-160 phospholipids, and then MSP is used as a hydrophobic group to bind to membrane proteins to maintain its natural conformation. A single membrane protein can form a nanodiscs of 150 kDa. When assembling nanodiscs, MSP was first mixed with phospholipids dissolved in detergents according to a certain proportion, then the detergents were removed by dialysis or hydrophobic adsorbents after incubation for a period of time, finally nanodiscs was self-assembled to form a nano-scale membrane-like structure disk. The self-assembly process of nanodiscs containing membrane proteins is similar to that of empty nanodiscs by adding appropriate amount of membrane proteins into the system. Nanodiscs containing membrane proteins and empty nanodiscs were separated by biochemical methods and then further purified by molecular exclusion chromatography.

Fig. 2 Self-assembly process of nanodiscs. (Vivien Yeh, 2018)

For the stable separation and purification of membrane proteins, the nanodiscs system can provide a membrane-like environment in solution, which has the same lipid composition as the cell membrane. Compared with other artificial membranes, the addition of MSP can better stabilize the natural conformation of membrane proteins. Like bicelles, the first step of nanodiscs is based on the solubilization of detergents, which needs to optimize the properties and concentration of detergents used, which may result in the loss of protein activity in nanodiscs recombination, so there are also restrictions on the use of detergents. Table 1 compares the composition, advantages and disadvantages of liposomes, bicelles and nanodiscs.

Table 1. Comparison of different types of artificial membrane.

Polymer Stabilization Strategy

In view of the deficiency of separation, purification and stabilization of membrane proteins by detergents and artificial membranes, the researchers developed a new method without adding detergents completely from the idea of protecting the local lipid environment around membrane proteins. That is, polymers are used to stabilize nano-scale lipid disks containing membrane proteins.

Styrene Maleic Anhydride (SMA) Polymer

SMA is a kind of copolymer with excellent properties obtained by free radical polymerization of styrene and maleic anhydride, which is alternately composed of hydrophilic maleic acid and hydrophobic styrene. SMA and about 140lipids compose styrene-maleic anhydride lipid particles (SMA lipid particles, SMALPs). At present, SMA method has been successfully used to stabilize and purify ion channel proteins, transporters, enzymes, respiratory chain complexes and receptors. When the ratio of styrene to maleic acid is 2:1 or 3: 1, SMA has the best effect on the separation and purification of membrane proteins. In addition, the extraction efficiency of SMA is also affected by the size of the target protein and the encapsulation density of the protein in the membrane. Compared with detergents and artificial membranes, SMA has the advantage of extracting target proteins with natural lipid bilayers and related proteins through spontaneous assembly of SMALPs. This not only provides information about the interaction between the relevant protein and the target protein, but also provides a method to identify the composition of endogenous lipids around the target protein. In addition, the membrane protein purified into SMALPs form had significant stability, and SMALPs could remain stable for at least one week at 4 ℃. After several rounds of freeze-thaw cycles, the loss of particle integrity and protein function of SMALPs was the least. However, the intolerance of SMA to low pH and divalent cations limits the application of this strategy in the downstream study of membrane proteins. Therefore, the structural modification of SMA to synthesize polymer lipid nanodiscs with adjustable pH value and tolerance to divalent cations is expected to be successfully applied to the stable extraction of membrane proteins.

Diisobutylene Maleic Acid (DIBMA) Polymer

The low tolerance of SMA to bivalent cations limits the use of cations such as Mg²⁺ and Ca²⁺, which are commonly used in buffers for protein purification and activity determination. Recent studies have shown that DIBMA copolymers can also directly dissolve the membrane to form DIBMA lipid particles (DIBMALPs), thus overcoming the limitations of SMA mentioned above. Oluwole et al. found that DIBMALPs is more tolerant to bivalent cations such as Mg²⁺ than SMALPs. Danielczak et al. found that when using DIBMA, adding Mg²⁺ or Ca²⁺ can improve the extraction efficiency. Although the purity and stability of DIBMA for some membrane proteins are lower than those of SMA, DIBMA has higher tolerance to bivalent cations, which provides a better environment for the study of protein conformation and kinetics. Therefore, the selection of polymers depends on the characteristics of the target proteins studied.

Polymethacrylate (PMA) Polymer

Although SMA and DIBMA have achieved success in the separation and purification of some membrane proteins, SMA has some defects such as low tolerance to divalent cations, low purity and stability of DIBMA extraction, and strong UV absorption of these two polymers, which may interfere with UV detection. Recently, amphiphilic PMA polymers have been shown to dissolve lipid bilayers into nanodiscs. PMA has potential advantages over common SMA polymers, but there are few applications of PMA in the purification of membrane proteins. Table 2 compares the composition, advantages, disadvantages and applications of SMA, DIBMA and PMA. Table 3 summarizes and compares the principle, classification, advantages and disadvantages of the above membrane protein stabilization techniques.

Table 2. Comparison of different types of polymers.

Table 3. Comparison of three membrane protein stabilization techniques.

Stabilization Reagents

Our membrane protein solubilization and stabilization reagents:

• Detergents Reagents
• Phospholipids Reagents
• Nanodiscs Reagents
• Polymer Reagents

Applications in Drug Screening

The screening of membrane protein ligand drugs and the discovery of biomarkers of membrane proteins are important directions in the research and development of new drugs. According to statistics, 54% of the approved drug targets in the DrugBank database are membrane proteins. The low expression and strong hydrophobicity of membrane proteins limit the in vitro study of membrane protein targets and the screening of ligand drugs. Therefore, the development of membrane protein stabilization technologies combined with surface plasmon resonance (SPR) technology has brought dawn to many potential membrane protein targets and related ligand drug screening to be developed.

At present, SPR is the most representative ligand high-throughput screening technology for membrane proteins. It is a sensitive surface analysis technique, which is detected by the change of dielectric constant caused by the adsorption of molecules on the heavy metal film. Since the 1990s, this method has been widely used in high-throughput screening of simple protein ligands and small molecular drugs. However, for multiple transmembrane proteins with low expression and complex structures, such as GPCRs and ABC transporters, membrane proteins usually lose function or denaturation when they come into direct contact with solid substrates, especially gold substrates in the Biacore system. Therefore, the researchers introduced the membrane protein stabilization technique to obtain membrane proteins with good activity, and then combined with SPR for the screening of their corresponding ligand drugs. Komolov et al. coupled the detergent stabilized rhodopsin protein to the surface of SPR chip and successfully used to analyze the interaction between rhodopsin protein and its ligand. Compared with the classical detergent strategy, artificial membrane stabilization strategy combined with SPR technology is more widely used in membrane protein ligand screening and new drug research and development. Das et al. combined nanodiscs stabilization technique with local surface plasmon resonance (LSPR) technique to analyze the binding types of 12 small molecular drugs to cytochrome P450 3A4 enzymes. In order to solve the limitation of GPCR ligand screening methods, Maynard et al. first used lipid bilayers to separate and purify GPCR, then immobilized it on the chip through nanopores, collected the dose-response curve of GPCR ligand binding by SPR technology, and established a high-throughput screening platform for GPCR ligands. Rich et al. established the best solubilization condition screening system of CCR5 protein by SPR technology. Using Fab (2D7) antibody as a positive drug, a detergent which can not only effectively dissolve the receptor but also maintain its activity was found among 96 different detergents. Xu et al. used nanodiscs-stabilized ion channel protein KcsA-Kv1 to couple to SPR chip to analyze the interaction between KcsA-Kv1 protein and its inhibitors.

Membrane protein stabilization technology is an indispensable key technology in drug screening for membrane proteins with complex structure. It can simulate the natural lipid environment needed by membrane proteins and ensure the integrity of the structure and function of membrane proteins to provide technical support for the establishment of high specificity, high sensitivity and high throughput drug screening methods.

Characterization Services

Our membrane protein characterization services:

• Membrane Protein Characterization
• Membrane Protein Characterization by Solution-state NMR Studies
• Membrane Protein Characterization by X-Ray Crystallography
• Membrane Protein Characterization by High-Throughput SPR and BLI Studies
• Membrane Protein Characterization by SAXS Studies
• Membrane Protein Characterization by Cryo-EM Studies
• Membrane Protein Functional Validation

What Can Creative Biolabs Do?

The functional integrity of membrane proteins is very important for the discovery of drug targets and the research and development of new drugs, and the stabilization technology of membrane proteins is the key to ensure their basic functional integrity. However, there are some technical difficulties in various membrane protein stabilization strategies, including how to increase the specificity of membrane protein extraction, how to reduce the loss of structure and function caused by the membrane protein extraction process, and the need for more comprehensive membrane protein functional verification to ensure the accuracy of established methods. Creative Biolabs can use a series of methods to express, purify, stabilize and characterize membrane proteins. With the efforts of our excellent R&D team, a series of new reagents have been used to provide a non-denaturing environment for the production of full-length membrane proteins with natural conformation and sufficient biological activity. Our customized membrane protein products perfectly meet the needs of antibody development, structural determination, functional and mechanism research, and other applications. With years of experience, we have achieved remarkable success in obtaining a variety of difficult membrane proteins such as G protein-coupled receptors (GPCR) ion channels membrane transporters and so on.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.