The Nature of Detergent and Its Application in Membrane Proteins
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In recent years, membrane protein research has made significant progress, which is inseparable from the development of membrane-related tools and reagents . Among them, the detergent plays an important role in the membrane protein extraction, purification and operation. Their amphiphilic nature allows them to interact with hydrophobic membrane proteins, extract and dissolve membrane proteins from natural lipid bilayers. But dissolution does not mean that the natural structure and stability of the protein can be completely restored; The detergent that can effectively extract the membrane protein at the same time may also not be suitable for purification and further biochemical studies; And the detergent that is suitable for a certain membrane protein may not be suitable for another membrane protein. In a word, there is no set of criteria that can estimate if a certain detergent is appropriate in the membrane protein study. This article describes the physical and chemical properties of detergents, as well as the application of detergent in membrane protein process, hope our introduction can help you to choose the right detergent.
1. Structure of Detergent
Detergent is a kind of surfactant, which has widely applications, including: polyacrylamide gel electrophoresis (PAGE), dissolution of inclusion bodies, preparation of liposomes, membrane protein solubilization and activity structure studies. Moreover, detergent can also be used as membrane model for in vitro studies.
The function of the detergent is related to its structure: The polar hydrophilic portion of the detergent molecule is used as a hydrophilic head group, while the non-polar hydrophobic portion is used as the tail (Figure 1A). Some detergents also have lenticular shapes (Figure 1B), which have polar and non-polar faces, including bile acid derivatives, such as CHAPS and CHAPSO.
Figure 1. Detergent monomer
2. Classification of Detergents
The detergents can be divided into ionic (cationic or anionic), nonionic and zwitterionic according to different hydrophilic groups.
The ionic detergents include sodium dodecyl sulfate (SDS), N-lauryl sarcosine, CTAB, etc., which are effective in extracting proteins from the membrane. These detergents can effectively disrupt the interaction between intramolecular and intermolecular proteins, but are harsh and tend to denature protein. Among them, bile acid salts are also ionic detergents, such as sodium cholate and deoxycholic acid, but their skeletons are composed of rigid steroids, which are milder than the linear ionic detergents.
Nonionic detergents include maltosides, glucosides and polyoxyethylene glycols. The feature of this type of detergent is that the hydrophilic head groups are not charged. These detergents are mild, non-denatured, and can disrupt interactions between protein-lipid and lipid-lipid.
The zwitterionic detergents include Zwittergents, Fos-Cholines, and CHAPS / CHAPSO, etc. Their hydrophilic head groups contain positive and negative charges. These detergents are electrically neutral like nonionic detergents, but they are usually able to disrupt the interaction between proteins like ionic detergents, so intermedium mild. Most successful NMR membrane proteins studies use the zwitterionic detergents, for example Fos-Choline 12.
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3. Physical Properties of Detergents
Micelles are aggregates of the detergent monomer in the solution, and the micelles forming process is called micellization . Detergent interacts with the membrane protein and membrane in the form of micelles, and the dissolution of the protein depends on the formation of micelles in the solution. Micelles are usually considered to have a "rough" surface, which is a dynamic structure. The detergent monomer in the micelles rapidly exchange with the free detergent monomer in the solution. Once the membrane protein is dissolved, we usually think that the detergent molecules form a torus around the hydrophobic transmembrane domain.
3.2 The Critical Micelle Concentration, CMC
The minimum concentration at which a detergent can form micelles is called the critical micelle concentration (CMC). Each kind of detergents has a CMC value, when the detergent concentration is higher than CMC, the monomer is self-assembled into non-covalent aggregate, also called micelles. The micellization does not actually occur at a single concentration, but occurs within a narrow concentration range.
When applying detergents to membrane proteins, one rule of thumb is that the working concentration of the detergent should be at least 2xCMC and the weight ratio of detergent to protein is at least 4: 1. When the membrane protein is dissolved from the original membrane, the working concentration of the detergent is much higher than that of CMC, and the molar ratio of detergent to lipid is 10: 1. Therefore CMC determines the amount of detergent to be added to various proteins and membrane products .
The CMC value of the detergent is not fixed, it will change with the pH, ionic strength and temperature of the solution . For example, the CMC value of the ionic detergent will decrease as the ionic strength increases.
3.3 Cloud Point
Cloud point refers to the temperature at which detergent solution is separated into two phases when the concentration is close to or higher than CMC. The micelle aggregates to form a turbid phase with high detergent concentration, and the detergent will be exhausted when the solution reaches the equilibrium point. The obtained two-phase solution can be separated, and the extracted protein is located in the phase rich in detergent. Because high cloud point temperature will cause denaturation of dissolved protein, it is recommended to use a detergent with low cloud point in the presence of protein. The cloud point is affected by factors such as changes in detergent concentration and temperature, and the addition of salts or polymers, such as dextran and polyethylene glycol. The cloud point is affected by factors such as changes in detergent concentration and temperature, and the addition of salts or polymers, such as dextran and polyethylene glycol.
4. The Application of Detergents in Membrane Proteins
4.1 Replacement or Removal of Detergents
Detergents that can effectively solubilize membrane proteins may be unsuitable for further biochemical studies, which require transferring membrane proteins to a more suitable detergent solution. When assembling liposomes or nanodiscs, we need to remove the detergent. The CMC can be used to determine the available method to replace or remove unwanted detergents. The detergents with high CMC are easily removed through dialysis, and the detergent solution can be diluted to a value below CMC through dialysis, then the micelles will break down into monomers and the monomers can easily pass through the dialysis membrane. In general, the detergent solution is dialyzed by detergent-free buffer with more than 200 times volumes, and change the buffer for several times in the middle period, for example Fos-Choline 12. The detergents with low CMC are typically removed by adsorption of hydrophobic beads, such as DDM. It can also be purified by nickel column. After the membrane protein binds to the column material, change the buffer with another detergent solution.
4.2 The Identification of Membrane Proteins
If the membrane protein is identified by SDS-PAGE, the boiling treatment may result in aggregation of membrane proteins. So we can incubate the membrane protein at room temperature for 10min, and then conduct gel electrophoresis. Membrane proteins usually do not migrate at the predicted molecular weight in SDS-PAGE. They generally migrate faster, that means their molecular weight will look smaller, probably because the fold is not complete or each molecular weight unit combine with more SDS than the water-soluble protein .
5. The Expression of Membrane Proteins
We just introduced the basic properties of detergents, but in fact the preparation of membrane protein is very difficult. For most of the membrane proteins, it's difficult to obtain sufficient quantity from the natural environment, therefore they need to be overexpressed. Unfortunately, it is difficult to obtain a sufficient amount of functional and stable membrane protein through E.coli or other expression systems. In general, the more transmembrane domains they have, the more difficult it is to express membrane proteins, such as aquaporins containing 6 transmembrane domains.
Aquaporin that is located at the cell membrane is a transmembrane protein with 6 transmembrane domains, and they form a "channel" on the cell membrane that can control the water in and out of the cell. The water molecules will form a single column when going through the aquaporin, when entering into the curved narrow channel, the internal dipolar force and polarity will help the water molecules to rotate at an appropriate angle through the narrow channel.
Aquaporins predominantly exist in mammalian kidneys and also exist in plants. Aquaporins play an important role in kidney urine concentration, digestive physiology, neurophysiology, respiratory physiology, eye physiology and skin physiology.
5.2 Active Aquaporins
Cusabio adopts E.coli cell free expression technology. The technology is not limited by cell structure and is suitable for the expression of membrane proteins and toxic proteins that are toxic to cells, with yield up to mg/ml level. According to the traditional cell-based expression, conventional treatment of membrane proteins requires destruction of the cell membrane, which tends to cause the therein inserted membrane conformation change or even denaturation. But the open E.coli cell free expression system can optimize expression yield in vitro in multiple ways, and the expressed membrane proteins can be immediately enveloped by the detergents after translation during the expression, to maximum avoid exposure to aqueous solution. Now we have already successfully developed the following active aquaporins.
● Recombinant Escherichia coli Aquaporin Z (aqpZ)
Function: Channel that permits osmotically driven movement of water in both directions. It is involved in the osmoregulation and in the maintenance of cell turgor during volume expansion in rapidly growing cells. It mediates rapid entry or exit of water in response to abrupt changes in osmolarity.
Figure 2. aqpZ in detergent micelles
Figure 3. The binding activity of aqpZ with ytfE
Activity: Measured by its binding ability in a functional ELISA. Immobilized aqpZ at 5 μg/ml can bind human ytfE.
The EC50 of human ytfE protein is 197.90-259.70 μg/ml.
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The next chapter 《The Application of Nanodiscs in Membrane Protein》
 R.M. Garavito, S. Ferguson-Miller, Detergents as tools in membrane biochemistry, J. Biol. Chem. 276 (2001) 32403–32406.
 Anatrace, Detergents and Their Uses in Membrane Protein Science.
 M. le Maire, P. Champeil, J.V. Mbller, Interaction of membrane proteins and lipids with solubilizing detergents, Biochim. Biophys.Acta 1508 (2000) 86–111.
 From Wikipedia, the free encyclopedia.
 Purifying Challenging Proteins Principles and Methods, 28-9095-31.