How do detergents get in hydrophobic membrane interior?

According to Molecular And Cellular Biology (Stephen L. Wolfe),

Membranes disperse almost instantaneously if exposed to a nonpolar environment or to detergents, which are amphipathic molecules that can form a hydrophilic coat around the hydrophobic portions of membrane lipids and proteins in water solutions.

This might be a stupid question but… if detergents can 'form coats around hydrophobic portions' of membrane-suspended molecules, they must, somehow get in the hydrophobic membrane interior… right?

How do they get in the membrane interior? Do they form clusters like endocytic vesicles? What happens after they form hydrophilic coats around hydrophobic molecule regions?

It depends on the concentration, but at higher concentration the detergent molecules build so called micelles, where the hydrophobic "tail" is orientation into the inner part and the hydrophilic "head" is orientated to the outside. This allows the micelle also to fuse with the membrane and then to desintegrate it. This illustration from the Wikipedia shows the schematically:

Its from the surfactant article, which has more details.

The membrane is dispersed by detergents. But the detergents, under the right concentrations and conditions (salt, pH, et) form micelles with smaller curvature than the lipids that compose cell membranes. With some luck, they can form a small hydrophobic micelle bubble around the protein.

This figure is depicts detergent micelles around a membrane protein's membrane bound regions, hopefully without distorting the shape of the protein!

Since you asked in the comments, here is a picture of the electron density of the detergent micelle in ompF Porin crystals - the detergent density is blue the protein is red. For a while in the mid 90s through maybe a few years ago there were a reasonable number of simulations including detergent molecules. The mobility of the chains in the micelles means that no specific model structure would be accurate, but it can help understand the electromagnetic environment of the solvent in cases which are important like charge transfer in a photoreaction center or the dynamics of transport through an import/export protein.

Hydrophobic effect

The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude water molecules. [1] [2] The word hydrophobic literally means "water-fearing", and it describes the segregation of water and nonpolar substances, which maximizes hydrogen bonding between molecules of water and minimizes the area of contact between water and nonpolar molecules. In terms of thermodynamics, the hydrophobic effect is the free energy change of water surrounding a solute. [3] A positive free energy change of the surrounding solvent indicates hydrophobicity, whereas a negative free energy change implies hydrophilicity.

The hydrophobic effect is responsible for the separation of a mixture of oil and water into its two components. It is also responsible for effects related to biology, including: cell membrane and vesicle formation, protein folding, insertion of membrane proteins into the nonpolar lipid environment and protein-small molecule associations. Hence the hydrophobic effect is essential to life. [4] [5] [6] [7] Substances for which this effect is observed are known as hydrophobes.

How do detergents get in hydrophobic membrane interior? - Biology

Here we present a comprehensive review of laboratory detergents and their applications in biomedical experiments. This review includes discussions of ionic, non-ionic and zwitterionic detergents, their general properties as well as information about commonly used detergents from each group. Finally, we include a brief discussion of Labome survey results for some common detergents.

Detergents used in biomedical laboratories are mild surfactants (surface acting agents), used for cell lysis (i.e., the disruption of cell membranes) and the release of intracellular materials. They are amphiphilic molecules, containing both hydrophilic and hydrophobic regions. This amphiphilic property allows detergents to break protein-protein, protein-lipid and lipid-lipid associations, denature proteins and other macromolecules, and prevent nonspecific binding in immunochemical assays and protein crystallization.

There are many types of detergents used in laboratory research. New amphiphilic compounds, usually designed for specific applications, continue to be developed (e.g., maltose-neopentyl glycol [1] and glycosyl-substituted dicarboxylates [2] ). This article reviews the characteristics and applications of the most commonly used laboratory detergents.

ionicsodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl
non-ionicTriton X-100, DDM, digitonin, tween 20, tween 80

Detergents are amphiphilic organic compounds comprised of a hydrophobic non-polar hydrocarbon moiety (tail) and a hydrophilic polar headgroup (Fig. 1A). This molecular structure is very similar to the amphiphilic phospholipids that make up our cellular membranes, except that the phospholipids possess pair hydrophobic tails attached to the hydrophilic headgroup (Fig 1D). When dissolved in water at appropriate concentrations and temperatures amphiphilic molecules self-assemble into structures that keep their hydrophilic headgroups on the exterior and the hydrophobic tails on the interior away from the water. Due to their molecular differences, detergent molecules form spherical micelles(Fig. 1C) while phospholipids are more likely to develop a bilayer (Fig 1D). The similarity in molecular structures allows the detergent to penetrate phospholipid bilayers and thus disrupt cell membranes.

Furthermore, the hydrophobic core of the micelle can bind to hydrophobic regions of proteins (Fig 1B). The number of detergent molecules in a micelle is called the aggregation number, an important parameter used to assess membrane protein solubility [3]. The length of the hydrophobic region is directly proportional to the degree of hydrophobicity, and it is quite constant among detergents, while the charged headgroup is variable. Both temperature and concentration are important parameters of phase separation and solubility of a detergent. The minimal detergent concentration at which micelles are observed at a given temperature is called the Critical Micelle Concentration (CMC). At any concentrations lower than the CMC, only monomers are observed at concentrations higher than CMC both micelles and monomers co-exist, along with other non-micellar phases that are not dissolved in water. Likewise, the lowest temperature at which micelles are formed is called Critical Micelle Temperature (CMT). CMC is also affected by the degree of lipophilicity of the headgroup. Generally, a low lipophilic or lipophobic character results in high CMC.

Common detergents are categorized into three groups based on their characteristics: ionic (anionic or cationic), non-ionic and zwitterionic. Below I discuss common detergents in each of these categories and provide important information about the selection and use of laboratory detergents.

Ionic detergents are comprised of a hydrophobic chain and a charged headgroup which can be either anionic or cationic. They generally have higher CMC values than non-ionic detergents and tend to be fairly harsh. Due to their charged headgroups, ionic detergents cannot be removed by ion exchange chromatography. Furthermore, additional precautions should be taken when using ionic detergents because some of their properties may be altered in buffers with variable ionic strength (e.g., CMC can fall dramatically when the NaCl concentration increases from 0 to 500 mM).

The anionic SDS is a very commonly used and effective surfactant in solubilizing most proteins. It disrupts non-covalent bonds within and between proteins, denaturing them, and resulting in the loss of their native conformation and function. SDS binds to a protein with a ratio of 1.4:1 w/w (corresponding to about one SDS molecule per two amino acids), masking the charge of the protein. Thus SDS adds an overall negative charge to all proteins in the sample regardless of their isoelectric point (pI). Once bound by negatively charged SDS molecules the proteins can be separated based on size. That is a big reason for the wide use of SDS polyacrylamide gel electrophoresis (SDS-PAGE) for separating and studying proteins. Usually, for complete cell lysis in the presence of SDS, a sample must be sonicated or sheared (e.g., passed through a 19G needle) several times to ensure DNA degradation. SDS cannot be used when active proteins are required or when protein-protein interactions are being studied because both of these are disrupted by the SDS. When working with SDS it is important to know that SDS precipitates at low temperatures, and this effect is enhanced in the presence of potassium salts. This phenomenon can sometimes be exploited to remove SDS from a protein sample [4].

Sodium deoxycholate and sodium cholate are bile salts detergents. They are both anionic detergents. These detergents are often used for membrane disruption and membrane protein extraction, for example, apelin receptor [5]. Deoxycholate does denature proteins while cholate is a non-denaturing detergent. One potential benefit to both of these detergents is that they can be removed from samples via dialysis, which may help with quantification and/or downstream analyses of proteins.

Sarkosyl, also known as sarcosyl or sodium lauroyl sarcosinate, is an anionic surfactant. It is amphiphilic due to the hydrophobic 14-carbon chain (lauroyl) and the hydrophilic carboxylate. The carboxylate with a pKa value of 3.6 is negatively charged in any physiological solution. Sarkosyl is prepared from lauroyl chloride and sarcosine in the presence of sodium hydroxide and is purified by recrystallization from alcohol, or by acidification with a mineral acid, separation of the free acid, and neutralization of the free acid. Sarkosyl has also been used to improve wetting and penetration of topical pharmaceutical products. In the food industry, sarkosyl is approved for use in processing, packaging, and transporting food for human consumption, and in adhesives used in food storage or transportation. It is widely used in cosmetic formulations such as shampoos and body washes at concentrations around 3-13% [6]. Sarkosyl is also used in metal finishing end processing for its crystal modifying, anti-rust, and anti-corrosion properties.

Sarkosyl is widely utilized in laboratory experiments, for example for solubilizing tau in Alzheimer disease research [7], due to its good water solubility, high foam stability, and strong sorption capacity to proteins. Sarkosyl serves as a detergent to permeabilize cells and extract proteins in isolation and purification techniques such as western blot and indirect ELISA. It can also inhibit the initiation of DNA transcription.

One major application of sarkosyl is for solubilizing and refolding proteins from inclusion bodies (protein aggregates within cytoplasm or nuclei). Eukaryotic recombinant proteins overexpressed in Escherichia coli tend to form such inclusion bodies. Sarkosyl is often used to solubilize an inclusion body pellet to extract the proteins and allow them to refold into their native form. Earlier work involved solubilizing inclusion bodies with denaturants, such as urea or guanidinium hydrochloride, and refolding by slow dilution — however, most of the solubilized proteins aggregate and precipitate upon removal of the strong detergents. Sarkosyl is an effective solubilizing agent that minimizes aggregation and allows refolding at higher protein concentrations (as much as 10-fold higher when compared to using guanidinium hydrochloride [8] ). One study found the over 95% of inclusion body fusion proteins were solubilized with 10% sarkosyl, and that the proteins could then be recovered with a mix of other detergents (i.e., Triton X-100 and CHAPS) [9]. Proteins in the soluble extract with sarkosyl can also be stored at 4°C for a week before affinity purification. It should be noted, however, that sarkosyl interferes with the subsequent chromatographic process and must be removed from the solution by dilution or dialysis.

Non-ionic detergents have uncharged hydrophilic headgroups. They are considered mild surfactants as they break protein-lipid and lipid-lipid associations, but typically not protein-protein interactions, and generally, do not denature proteins. Therefore, many membrane proteins may be solubilized in their native and active form, retaining their protein interactors. However, because not all proteins behave the same with different non-ionic detergents, trial and error may be necessary to find the best detergent for your protein(s) of interest. Additionally, it should be noted that most non-ionic detergents interfere with ultra-violet (UV) spectrophotometry. Therefore, protein determination at 280 nm in the presence of non-ionic detergents is typically imprecise.

All members of the Triton family: Triton X-100, Triton X-114, Nonidet P-40 (NP-40), Igepal® CA-630, are quite similar, differing slightly in their average number (n) of monomers per micelle (9.6, 8.0, 9.0, and 9.5, respectively) and the size distribution of their polyethylene glycol (PEG)-based headgroup. The CMC values of these detergents are low, and therefore they can not be easily removed by dialysis. Triton X-100, a typical non-ionic detergent, derives from polyoxyethylene and contains an alkylphenyl hydrophobic group. Triton X-100 is commonly used for isolating membrane protein complexes, and the surfactant of choice for most such as for co-immunoprecipitation experiments. Other members of the Triton family are used for membrane protein isolation by phase-separation due to low cloud points (the temperature at which the micelles aggregate and form a distinct phase). While the cloud point of Triton X-100 is 64°C, the cloud point of Triton X-114 is 23°C. This allows for membrane protein extraction and solubilization in Triton X-114 without bringing the samples up to warmer temperatures which may denature many proteins.

Brij™ 35 is another nonionic polyoxyethylene surfactant, commonly used as a component of cell lysis buffers or assay buffers or a surfactant in HPLC applications.

The n-dodecyl-β-D-maltoside (DDM) is a glycosidic surfactant, increasingly used with hydrophobic and membrane protein isolation when the protein activity needs to be preserved. It is more efficient at protein solubilization for 2-D electrophoresis than several other detergents, including CHAPS and NP-40 [10]. The glycochain in its lipophilic site, its high CMC of 0.17 mM and the interface of the micelles create an aqueous-like microenvironment ideal for solubilizing and retaining the stability of membrane and hydrophobic proteins [11]. For example, Winkler MBL et al purified NCR1 protein with the addition of n-dodecyl-β-D-maltopyranoside [12] so did Li Y et al for LptB2FG and LptB2FGC proteins [13]. Steichen JM et al mixed protein complexes in a solution of DDM from Anatrace before Cryo-EM [14].

Other maltosides, such as beta-decyl-maltoside, have different lengths of the hydrophobic alkyl chains. Glucoside (octyl-glucoside) are a potential alternative to maltoside detergents for protein research [15].

Digitonin, a steroidal glycoside derived from the purple foxglove plant (Digitalis purpurea), is used for the solubilization of cellular membranes. As with other non-ionic detergents discussed here, digitonin is frequently used to solubilized membrane proteins without denaturing them. For example, B de Laval et al lysed cells with 1% of digitonin for Tn5 transposase reaction during the ATAC-seq protocol [16]. Zhao Y et al released synaptic and extrasynaptic AMPA receptors from postsynaptic density with digitonin [17]. Additionally, digitonin is used to extract cellular organelles. Digitonin interacts with cholesterol in membranes and thus can be used to permeabilize the cholesterol-rich plasma membrane while leaving the cholesterol-poor organelle membranes intact.

Tween-20 and Tween-80 are polysorbate surfactants with a fatty acid ester moiety and a long polyoxyethylene chain. They have very low CMC, are generally gentle surfactants, do not affect protein activity and are effective in solubilization. Tweens are not common ingredients of cell lysis buffers however, they are routinely used as washing agents in immunoblotting and ELISA to minimize nonspecific binding of antibodies and to remove unbound moieties, and used to permeabilize cell membranes. For example, Yang J et al immunostained intracellular FLAG tag after treating HEK293 cells with 0.2% Tween 20 [18].

One common question regarding the Tween family detergents is the difference between Tween 20 and Tween 80, the two most commonly used members. Tween 20 has lauric acid, while Tween 80 has oleic acid (Figure 3). Table 2 summarizes various aspects between them. These detergents can often be used interchangeably however, the difference between them is sometimes important, such as in in vivo studies that may be influenced by the different levels of hemolytic effect of Tween 20 and Tween 80 [19]. Greenwood DJ et al, for example, grew Mycobacterium tuberculosis in a medium supplemented with 0.05% Tween 80 [20]. Ouadah Y et al injected a dibenzazepine solution with 0.1% v/v Tween 80 into mice to inhibit Notch signalling [21].

Synonyms Chemical Formula Molecular Weight Density (g/mL) Appearance Applications
Tween 20polysorbate 20, polyoxyethylene sorbitan monolaurate, PEG (20) sorbitan monolaurateC 58 H 114 O 26 12281.1Clear, yellow to yellow-green viscous liquida broad range of applications: as a blocking agent in PBS or TBS wash buffers for ELISA, Western blotting and other immunoassay methods for lysing mammalian cells and as a solubilizing agent for membrane proteins.
Tween 80polysorbate 80, polyoxyethylene sorbitan monooleate, PEG (80) sorbitan monooleateC 64 H 124 O 26 13101.06-1.09amber colored viscous liquidas a stabilizing agent for proteins used in tests for the identification of phenotype of some mycobacteria used in vaccine preparations [22]

Though non-ionic detergents are generally relatively mild, many proteins do denature or aggregate in the presence of these detergents. To ameliorate this issue new non-ionic glyco-lithocholate amphiphiles (GLC-1, GLC-2, and GLC-3) and glyco-diosgenin amphiphile (GDN) have been developed [23]. GDN was used to extract yeast mitochondrial dimeric ATP synthase to understand better how the protein functions [24]. Pluronic F-68 is commonly used in suspension cell culture at 0.1% to reduce the water shear force [25].

The headgroups of zwitterionic detergents are hydrophilic and contain both positive and negative charges in equal numbers, resulting in zero net charge. They are more harsh surfactants than the non-ionic detergents. A typical zwitterionic detergent is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, better known as CHAPS. CHAPS high CMC (6 mM at room temperature) allows efficient removal by dialysis. It is very common in sample preparation at concentrations of 2-4% for isoelectric focusing and 2D electrophoresis. CHAPSO differs with CHAPS in that it contains a more polar headgroup, which makes it more capable of solubilizing hydrophobic molecules. Thus, CHAPSO is mainly used for solubilization of integral membrane proteins.

Chaotropic agents are similar substances to surfactants in that they break non-covalent interactions (hydrogen bonds, dipole-dipole interactions, hydrophobic interactions) facilitating protein denaturation, which in this case is usually reversible. Urea is a common chaotropic agent used alone, or in combination with thiourea or other detergents, in applications like 2D-gel electrophoresis and in-solution enzymatic digestion of proteins for preparation during proteomic workflows. When using Urea, extra care must be taken not to heat the sample above 37°C as this will lead to carbamylation of proteins [26].

For membrane protein solubility, a detergent with high CMC should generally be chosen, and the volume and concentration of the buffer are also crucial as enough detergent should be present to solubilize all membrane proteins in the sample. In most cases, the detergent concentration should be well about the CMC level (at least 2X the CMC) to ensure sufficient micelle concentration to solubilize the membrane proteins. According to Linke [3], at least one micelle is needed per membrane protein molecule to sufficiently mimic the lipid environment of a membrane (Fig. 1B, D).

Phase separation can be used to purify the proteins further. This requires adjusting the temperature and the concentrations of salts and detergent in the buffer to cause the detergent micelles to aggregate and separate from the aqueous layer. In this case, the membrane proteins, surrounded by the micelles, aggregate with the detergent. The temperature at which the detergent solution separates into two phases, the cloud point, is affected by glycerol or salts in the buffer (e.g., Triton X-114 has a cloud point of 23°C, but in the presence of 20% glycerol, the cloud point declines to 4°C). This is very important since the stability of a protein is affected by high temperatures.

A good detergent should be able to lyse cells, solubilize proteins and be suitable for your downstream application(s). Also, the solubilized protein in native or denatured form should be considered. There is no ideal detergent for all applications, and even in the same application, the result varies (Table 3). Therefore, after options are considered, trial and error are often necessary to find the best detergent, and a mixture of detergents may be optimal. Also, the fresh preparation of detergent working solution is usually the best practice to avoid hydrolysis and oxidation.

DetergentMW (Da) monomerMW (Da) micelleCMC (mM) 25 o CAggregation No.Cloud Point ( o C)Avg. Micellar WeightStrengthDialyzableApplications
SDS28918,0007-1062>10018,000HarshYesCell lysis, Electrophoresis, WB, hybridization
Triton X-10062590,0000.2-0.9100-1556580,000MildNoEnzyme immunoassays, IP, Membrane solubilization
CHAPS6156,150610>1006,150MildYesIEF, IP
NP-4068090,0000.059 45-50 MildNoIEF
n-dodecyl-β-D-maltoside511 0.1598 50,000 Protein Crystallization
Tween-201228 0.06 76 MildNoWB, ELISA, Enzyme immunoassays
Digitonin122970,000<0.560 70,000MildNoMembrane solubilization

The downstream applications often require that detergent concentrations be lowered or completely removed. For such purposes, size exclusion chromatography or dialysis can be used if the micelle size is substantially different than the protein of interest or micelles are small enough (i.e., high CMC) to pass through the dialysis tubing [3]. Other methods employ the use of detergent binding non-polar beads or resins, cyclodextrin inclusion compounds [27], ion-exchange chromatography or protein precipitation. However, the buffer used after detergent removal must be selected carefully to avoid protein precipitation or aggregation.

Labome surveys the literature for the application of detergents. The following table lists the main suppliers, and the number of articles, indicating most of the detergents are supplied by MilliporeSigma.

detergent suppliers
Triton X-100Thermo Fisher [28, 29], Electron Microscopy Sciences [30], Amresco, JT Baker
Tween-20Bio-Rad [31], MilliporeSigma [29], Thermo Fisher
SDSAmresco, Bio-Rad, Q.BIOgene, MilliporeSigma
NP-40Roche, MilliporeSigma [32]
CHAPSMilliporeSigma, JT Baker
digitoninMilliporeSigma, Wako
DDMGeneron [33], Anatrace [15]

Thermo Fisher Pierce Triton X-100, for exampl, BP151 [29] or 85111 [28], was used to lyse cell and tissue samples for immunohistochesmitry [29] and immunocytochemistry [28]. MilliporeSigma Triton X-100 was used to lyze cells [34], or permeabilize cells in immunocytochemistry [35], and in blocking buffer for immunohistochemistry [36, 37] and proteinase K protection assay [38].

Tween-20 is commonly used in washing buffers, such as TBS-Tween (TBS-T) or PBS-Tween (PBT-T), in various immunoassays. MilliporeSigma Tween-20, for example, P1379 [29], was used in washing blots [29], in IHC experiments (P1379) [39], in immunoprecipitation [40],and in microfluidic array multiplex PCR [41] and others [42]. MilliporeSigma Tween-80, was used to dissolve erlotinib (a chemotherapy drug) [43] and as a supplement to grow M. tuberculosis strains [44].

Lonza SDS (catalog number 51213) was used in chromatin preparations [39]. Amresco SDS was used in SDS-PAGE [45]. Bio-Rad sodium dodecyl sulfate was used to prepare a radioimmunoprecipitation assay buffer [46]. MilliporeSigma-Aldrich SDS was used to prepare buffers for, among others, in vitro octanoylation assays, Laemmli sample buffer, 2D-DIGE experiments [47].

Roche NP-40 was used in cell lysis [48, 49]. MilliporeSigma NP-40 was used to prepare radioimmunoprecipitation assay buffer [46], cell lysis/homogenization buffers buffer [50, 51] and immunoprecipitation assay RIPA buffer [52].

MilliporeSigma CHAPS was used in buffers for protein crystallization [53]. JT Baker CHAPS was used to lyse cells to study viral interaction with human ASF1 protein [54].

MilliporeSigma was used in an immunocytochemistry experiment to study PI4P [55] and used to perform proteinase K protection assays [38], and to extract RNA [56]. Wako digitonin was used to lyse cells [57] and perform immunoprecipitation experiments [58].

Y Lee et al solubilized a GPCR protein with dodecylmaltoside / DDM from Generon [33]. Anatrace n-decyl-beta-D-maltopyranoside was used for protein purification [59, 60] so were its n-dodecyl-beta-D-maltoside [61] and n-undecyl-beta-D-maltoside [62, 63]. Glycon beta-dodecyl-maltoside and beta-decyl-maltoside were also used in protein purification [64]. Anatrace n-octyl-beta-glucoside was used in solubilizing AQP4 proteins [15].

Silva MC et al used Brij-35 as the detergent for bio-layer interferometry biosensor assay [65]. For protein purifications, Affymetrix octyl glucose neopentyl glycol (OGNPG) at 1% [66], and MilliporeSigma cholesteryl hemisuccinate at 0.1% or 0.05% (w/v) [11, 67] were used. For chromatin-related assays, MilliporeSigma-Aldrich sodium deoxycholate (catalog number D6750) and Igepal (catalog number I8896), and TEKnova N-lauroylsarcosine (catalog number S3379) were used [39].

Detergents for Cell Lysis and Protein Extraction

Detergents are amphipathic molecules, meaning they contain both a nonpolar "tail" having aliphatic or aromatic character and a polar "head". Ionic character of the polar head group forms the basis for broad classification of detergents they may be ionic (charged, either anionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero).

Detergents in solution

Like the components of biological membranes, detergents have hydrophobic-associating properties as a result of their nonpolar tail groups. Nevertheless, detergents are themselves water-soluble. Consequently, detergent molecules allow the dispersion (miscibility) of water-insoluble, hydrophobic compounds into aqueous media, including the extraction and solubilization of membrane proteins.

Detergents at low concentration in aqueous solution form a monolayer at the air–liquid interface. At higher concentrations, detergent monomers aggregate into structures called micelles. A micelle is a thermodynamically stable colloidal aggregate of detergent monomers wherein the nonpolar ends are sequestered inward, avoiding exposure to water, and the polar ends are oriented outward in contact with the water.

Idealized structure of a detergent micelle.

Both the number of detergent monomers per micelle (aggregation number) and the range of detergent concentration above which micelles form (called the critical micelle concentration, CMC) are properties specific to each particular detergent (see table). The critical micelle temperature (CMT) is the lowest temperature at which micelles can form. The CMT corresponds to what is known as the cloud point since detergent micelles form crystalline suspensions at temperatures below the CMT and are clear again at temperatures above the CMT.

Detergent properties are affected by experimental conditions such as concentration, temperature, buffer pH and ionic strength, and the presence of various additives. For example, the CMC of certain nonionic detergents decreases with increasing temperature, while the CMC of ionic detergents decreases with addition of counter ion as a result of reduced electrostatic repulsion among the charged head groups. In other cases, additives such as urea effectively disrupt water structure and cause a decrease in detergent CMC. Generally, dramatic increases in aggregation number occur with increasing ionic strength.

Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking protein–protein interactions. Non-denaturing detergents can be divided into nonionic detergents such as Triton X-100, bile salts such as cholate, and zwitterionic detergents such as CHAPS.

Properties of common detergents.

Thermo Scientific Triton X-100Nonionic140647 (90K)0.24 (0.0155)64No
Thermo Scientific Triton X-114Nonionic537 ( – )0.21 (0.0113)23No
NP-40Nonionic149617 (90K)0.29 (0.0179)80No
Thermo Scientific Brij-35Nonionic401225 (49K)0.09 (0.0110)>100No
Thermo Scientific Brij-58Nonionic701120 (82K)0.08 (0.0086)>100No
Thermo Scientific Tween 20Nonionic1228 ( – )0.06 (0.0074)95No
Thermo Scientific Tween 80Nonionic601310 (76K)0.01 (0.0016)No
Octyl glucosideNonionic27292 (8K)23-24 (

‡Agg.# = Aggregation number, which is the number of molecules per micelle.

Purified detergent solutions

Although detergents are available from several commercial sources and used routinely in many research laboratories, the importance of detergent purity and stability is not widely appreciated. Detergents often contain trace impurities from their manufacture. Some of these impurities, especially peroxides that are found in most nonionic detergents, will destroy protein activity. In addition, several types of detergents oxidize readily when exposed to the air or UV light, causing them to lose their properties and potency as solubilizing agents. We offer several high purity, low peroxide–containing detergents that are packaged under nitrogen gas in clear glass ampules. These Thermo Scientific Surfact-Amps Detergent Solutions provide unsurpassed convenience, quality and consistency for all detergent applications. A sampler kit includes 10 different purified detergents (seven in the Surfact-Amps format and three in solid form).

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Structure of cell membranes

A major factor determining the behavior and interaction of molecules in biological samples is their hydrophilicity or hydrophobicity. Most proteins and other molecules with charged or polar functional groups are soluble (or miscible) in water because they participate in the highly ordered, hydrogen-bonded intermolecular structure of water. Some other proteins (or at least parts of proteins), as well as fats and lipids, lack polar or charged functional groups consequently, they are excluded from the ordered interaction of water with other polar molecules and tend to associate together in structures having minimal surface area contact with the polar environment. This association of nonpolar molecules in aqueous solutions is commonly called hydrophobic attraction, although it is more accurately understood as exclusion from the hydrophilic environment.

The formation and stability of biological membranes results in large measure from the hydrophobic attraction of phospholipids, which form bilayer sheets having hydrophobic lipid "tails" oriented within the sheet thickness and polar "head" groups oriented to the outer and inner aqueous environments. Membrane proteins completely span the membrane thickness or are embedded at one side of the membrane in accord with their structure of hydrophobic and hydrophilic amino acid side chains and other functional groups.

Membrane disruption, protein binding and solubilization

Generally, moderate concentrations of mild (i.e., nonionic) detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Using certain buffer conditions, various detergents effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins.

Detergent-based cell lysis. Both denaturing and non-denaturing cell lysis reagents may be used for protein extraction procedures.

Denaturing detergents such as SDS bind to both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins at concentrations below the CMC (i.e., as monomers). The reaction is equilibrium driven until saturated. Therefore, the free concentration of monomers determines the detergent concentration. SDS binding is cooperative (i.e., the binding of one molecule of SDS increases the probability that another molecule of SDS will bind to that protein) and alters most proteins into rigid rods whose length is proportional to molecular weight.

Non-denaturing detergents such as Triton X-100 have rigid and bulky nonpolar heads that do not penetrate into water-soluble proteins consequently, they generally do not disrupt native interactions and structures of water-soluble proteins and do not have cooperative binding properties. The main effect of non-denaturing detergents is to associate with hydrophobic parts of membrane proteins, thereby conferring miscibility to them.

At concentrations below the CMC, detergent monomers bind to water-soluble proteins. Above the CMC, binding of detergent to proteins competes with the self-association of detergent molecules into micelles. Consequently, there is effectively no increase in protein-bound detergent monomers with increasing detergent concentration beyond the CMC.

Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization. The initial stage is lysis or rupture of the membrane. At detergent:membrane lipid molar ratios of 0.1:1 through 1:1, the lipid bilayer usually remains intact but selective extraction of some membrane proteins occurs. Increasing the ratio to 2:1, solubilization of the membrane occurs, resulting in mixed micelles. These include phospholipid–detergent micelles, detergent–protein micelles, and lipid–detergent–protein micelles. At a ratio of 10:1, all native membrane lipid:protein interactions are effectively exchanged for detergent:protein interactions.

The amount of detergent needed for optimal protein extraction depends on the CMC, aggregation number, temperature and nature of the membrane and the detergent. The solubilization buffer should contain sufficient detergent to provide greater than 1 micelle per membrane protein molecule to help ensure that individual protein molecules are isolated in separate micelles.

Detergents used for cell lysis. Major characteristics of denaturing and non-denaturing detergents used for protein extraction.

How Detergents Work

Neither detergents nor soaps accomplish anything except binding to the soil until some mechanical energy or agitation is added into the equation. Swishing the soapy water around allows the soap or detergent to pull the grime away from clothes or dishes and into the larger pool of rinse water. Rinsing washes the detergent and soil away.

Warm or hot water melts fats and oils so that it is easier for the soap or detergent to dissolve the soil and pull it away into the rinse water. Detergents are similar to soap, but they are less likely to form films (soap scum) and are not as affected by the presence of minerals in the water (hard water).

Content Background: How Does an Anabolic Steroid Reach its Target?

Once in the bloodstream, the anabolic steroid travels to all tissues in the body, where it enters the cells to reach its target. In order to get into a muscle cell for example, the steroid must leave the capillary and then enter the muscle cell. This means that the steroid must cross two different types of membranes, the capillary membrane and the muscle cell membrane. To cross the capillary membrane, there are numerous pores or fenestra 1 , which allow small molecules to squeeze through (Figure 3 and see Module 1). However the muscle cell membrane (like most cells in the body) does not have these small pores and therefore the steroid can only cross the membrane by diffusing across or by transport via a carrier protein. Steroids cross the cell membrane by passive diffusion 2 , which occurs in the direction of the concentration gradient – this does not require energy. Passive diffusion depends on the physiochemical characteristics of the membrane and the drug 3 . The muscle cell membrane, like all cell membranes in the body, is a lipid bilayer (Figure 4). It consists of lipids arranged with their polar 4 head groups facing the outside and inside of the cell. The chains of fatty acids face each other, forming the hydrophobic 5 (water-fearing) or non-polar 6 interior. Because anabolic steroids 7 are very lipophilic 8 (lipid-loving), they diffuse easily into the hydrophobic membrane interior. As they concentrate within the hydrophobic membrane interior, a new driving force is generated, pushing the steroid into the cytoplasmic side of the cell membrane. Once the anabolic steroid diffuses into the cytoplasm of the cell, it binds to the androgen receptor 9 (Figure 5). [Receptors for other steroids are found in the nucleus instead of the cytoplasm.] This complex of steroid and protein then crosses the nuclear membrane to enter the nucleus of the cell, where it exerts its effects. In this case, passive diffusion can’t occur because the protein is too large and not lipophilic. Instead, the steroid-receptor complex moves through small pores in the nuclear membrane to enter the nucleus. Although scientists are still elucidating exactly how this occurs, it is possible that the complex interacts with transport proteins that line the nuclear pores. This is an example of facilitated diffusion 10 , which occurs in the direction of the concentration gradient. Therefore, no energy is required. This is unlike active transport 11 , which occurs against the concentration gradient, and requires energy.

1 small spaces or pores within endothelial cells that form the capillary membrane. These pores allow charged drugs or larger drugs to pass through the capillaries.
2 the movement of a solute in its uncharged form to cross a membrane along a concentration gradient. No energy is required.
3 a substance that affects the structure or function of a cell or organism.
4 a chemical property of a substance that indicates an uneven distribution of charge within the molecule. A polar substance or drug mixes well with water but not with organic solvents and lipids. Polar or charged compounds do not cross cell membranes (lipid) very easily.
5 “water-fearing” a compound that is soluble in fat but not water. This is typical of compounds with chains of C atoms.
6 a chemical property of a substance that indicates an even distribution of charge within the molecule. A non-polar or non-charged compound mixes well with organic solvents and lipids but not with water.
7 synthetic versions of testosterone designed to promote muscle growth without producing androgenic effects. The better term is anabolic-androgenic steroid.
8 high lipid solubility. Lipophilic compounds dissolve readily in oil or organic solvent. They exist in an uncharged or non-polar form and cross biological membranes very easily.
9 a protein to which hormones, neurotransmitters and drugs bind. They are usually located on cell membranes and elicit a function once bound.
10 the movement of molecules across a membrane with the concentration gradient. No energy is required, but transport proteins can become saturated, limiting the diffusion process.
11 the movement of molecules against the concentration gradient with the help of a transport protein. This transport requires energy in the form of ATP.

Figure 3 A capillary is composed of endothelial cells that connect together loosely. Small pores or fenestrae are also present, allowing solutes to move in and out of the capillaries.

Figure 4 Schematic view of cell membrane. Lipids are arranged with polar head-groups facing the outside and inside of the cell, while the fatty acid chains form the non-polar (hydrophobic) membrane interior.

Figure 5 Testosterone (or anabolic-androgenic steroids) binds to the androgen receptor in the cytoplasm and the complex moves into the nucleus where it interacts with DNA to initiate protein synthesis.


Detergents are critical tools for the study of membrane proteins. They are vital for the isolation and purification of the proteins and are used in the primary solubilization step of reconstitution. They are invaluable in membrane protein recrystallization.

So What are detergents? Detergents are soluble amphiphilic molecules consisting of a polar head group and hydrophobic chain (or tail) and exhibit unique properties in aqueous solutions in which they spontaneously form spherical micellar structures. Membrane proteins are frequently soluble in micelles formed by amphiphilic detergents. Detergents solubilize membrane proteins by creating a mock lipid bilayer environment normally inhabited by the protein.

3 Major Detergent Classifications

    • Ionic Detergents: These have a polar head that can be either anionic or cationic and a hydrophobic chain or tail with a steroidal backbone. They are very efficient at solubilizing proteins, but almost always cause denaturation of the protein to some extent. An example of an ionic detergent is Sodium Dodecyl Sulfate (SDS).

      • Non-Ionic Detergents: These detergents have an uncharged hydrophilic head of either Polyoxyethylene or glycosidic group. It is a relatively mild detergent that solubilizes proteins by breaking the lipid-lipid interactions or lipid-protein interactions. Ionic detergents do not break the protein-protein interactions thereby, the solubilized protein is structurally intact in its biological form. Ionic detergents are effective in isolating active membrane proteins. Examples of Non-Ionic detergents are - n-Octyl-β-D-glucopyranoside (OG) and n-dodecyl-β-D-maltoside(DDM).
      • Zwitterionic Detergents: The polar head groups of zwitterionic detergents have a neutral charge. They have both ionic and non-ionic properties. The strength of action of zwitterionic detergents is intermediate between both ionic and non-ionic detergents.

      Zwitterionic Detergents for Membrane Proteins

      Zwitterionic detergents are efficient at breaking protein-protein interactions and are less harsh than ionic detergents. While zwitterionic detergents break the protein bonds, they are still successful at maintaining the native state and charge of individual proteins.
      Due to their versatility, Zwitterionic detergents are useful in a variety of applications such as:

      • Chromatography
      • Different types of electrophoresis, including 2D gel electrophoresis
      • Mass spectrometry
      • Solubilization of organelles and inclusion bodies.

      Examples of zwitterionic detergents include CHAPS and CHAPSO.

      Synthetic zwitterionic detergents are known as sulfobetaines. This group of substances retain their zwitterionic characteristics over a wide pH range.

      YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis

      Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN W279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

      Keywords: bacterial pathogenesis molecular modeling mutagenesis protein secretion protein-protein interaction regulation.


      32 kDa) polypeptide, while the double asterisk ( ** ) reveals the naturally produced and secreted

      42 kDa YopN-TyeA hybrid. The arrows (←) indicate a non-specific protein band recognized by the anti-YopN antiserum and the anti-YopD antiserum. The band appearing just above the nonspecific band in the ΔtyeA strain likely represents a frameshifting event that causes full-length YopN to be fused with the TyeAΔ19−59 deletion remnant resulting in a hybrid product that has a predicted molecular weight of

      38 kDa. Strains: Parent (YopNnative), YPIII/pIB102 ΔyscU, lcrQ double mutant, YPIII/pIB75-26 ΔyopN null mutant, YPIII/pIB82 ΔtyeA null mutant, YPIII/pIB801a ΔyopN, tyeA double mutant, YPIII/pIB8201a Mutant 1–YopN288(scramble)293, YPIII/pIB8213 Mutant 2–YopN288STOP, YPIII/pIB8212 Mutant 3–YopN279(F+1), 287(F−1), YPIII/pIB8208 Mutant 4–YopN279(F+1), 287STOP, YPIII/pIB8207 Mutant 5–YopN279STOP, YPIII/pIB8209. The theoretical molecular masses predicted from amino acid sequence are given in parentheses.

      How do detergents get in hydrophobic membrane interior? - Biology

      Detergents are invaluable tools for studying membrane proteins. However, these deceptively simple, amphipathic molecules exhibit complex behavior when they self-associate and interact with other molecules. The phase behavior and assembled structures of detergents are markedly influenced not only by their unique chemical and physical properties but also by concentration, ionic conditions, and the presence of other lipids and proteins. In this minireview, we discuss the various aggregate forms detergents assume and some misconceptions about their structure. The distinction between detergents and the membrane lipids that they may (or may not) replace is emphasized in the most recent high resolution structures of membrane proteins. Detergents are clearly friends and foes, but with the knowledge of how they work, we can use the increasing variety of detergents to our advantage.

      Published, JBC Papers in Press, June 29, 2001, DOI 10.1074/jbc.R100031200

      This minireview will be reprinted in the 2001 Minireview Compendium, which will be available in December, 2001. This is the third article of four in the “Membrane Protein Structural Biology Minireview Series.” Some of the work discussed in this minireview was supported in part by National Institutes of Health Grants P01 GM57323 (to R. M. G. and S. F. M.) and HL56773 (to R. M. G.).

      This minireview is dedicated to Drs. Jacqueline A. Reynolds and the late Martin Zulauf who gave one of us (R. M. G.) invaluable insights into the behavior of detergents.

      To whom correspondence may be addressed. Tel.: 517-355-9724 Fax: 517-353-9334 E-mail: [email protected]

      To whom correspondence may be addressed. Tel.: 517-355-0199 Fax: 517-353-9334 E-mail: [email protected]

      Modeling Protein–Protein and Protein–Nucleic Acid Interactions: Structure, Thermodynamics, and Kinetics

      Huan-Xiang Zhou , . Harianto Tjong , in Annual Reports in Computational Chemistry , 2008

      3.1 Electrostatic contribution

      It is well understood that hydrophobic interactions make favorable contributions to binding. However, the effects of electrostatic interactions are subtle. Neglecting conformational changes, the electrostatic contribution is given by

      where G el is the electrostatic free energy of each subunit (A or B) or the complex (AB), which can be calculated by solving the Poisson–Boltzmann (PB) equation. The subtlety of the electrostatic contribution can be appreciated by decomposing it into two components: the desolvation cost W desol and the solvent-screened interaction energy W int ( Figure 4.2 ). To obtain W desol , the electrostatic solvation energy of each subunit is calculated twice, first by itself and then in the presence of its partner, which has its partial charges zeroed out. The difference in the results between these two calculations gives the desolvation cost for that subunit, and adding the corresponding quantity for its partner gives W desol . The difference between W el and W desol comes from the interactions between the partial charges of the two subunits in the solvent environment.

      Figure 4.2 . Decomposition of the electrostatic contribution to binding affinity into desolvation cost and solvent-screened interaction. Interactions of protein charges with the solvent (represented by shadows around binding molecules) are indicated by outgoing arrows. Upon binding, the binding molecules are desolvated within their interface and charge–charge interactions, as indicated by a double-headed arrow, emerge.

      It is clear that W desol opposes binding. W int will favor binding when the charges on the two subunits have complementary charge distributions, which should be true in general. There W el consists of two opposite components. Whether electrostatic interactions make a net positive or net negative contribution to binding rests on the balance between the two components. In particular, the balance is very sensitive to how the boundary between the protein low dielectric and the solvent high dielectric is precisely specified. As shown on a large number of protein–protein and protein–RNA complexes [16–19] , when the dielectric boundary is chosen as the molecular surface (MS), as is often done in the literature, W desol outweighs W int , leading to net destabilization. However, when the dielectric boundary is switched to the van der Waals (vdW) surface, the situation is reversed and electrostatic stabilization is now predicted.

      How can one then decide on the choice of the dielectric boundary? One possibility is to benchmark PB calculations against explicit-solvent molecular dynamics (MD) simulations. Most of such efforts have been limited to small solute molecules [20–22] . However, it has been shown that the difference between MS and vdW results for electrostatic solvation energies depends on solute size [23] . Therefore parameterization on small solutes (either against explicit-solvent MD results or against experimental data) may not be reliable for calculating electrostatic contributions to protein–protein and protein–nucleic acid binding.

      One can benchmark PB calculations directly against experimental data on protein–protein and protein–nucleic acid binding affinities. Potentially one type of useful data is the dependence of binding affinities on salt concentration. The screening of electrostatic interactions by salts can be captured by the PB equation (it should be mentioned that salts can also specifically bind to proteins and nucleic acids such specific salt effects require special treatment). Unfortunately, it has been found that the screening effects predicted by MS and vdW calculations are essentially identical and thus cannot discriminate between the two choices of the dielectric boundary [16,18] . On the other hand, effects of mutations involving charged or polar residues have been found to have discriminating power, with experimental data favoring the vdW surface as the choice for the dielectric boundary [16–18] . Experimental data for mutational effects on binding affinity continue to accumulate in the literature [24,25] , providing opportunities for comprehensive benchmarking of PB calculation protocols.

      In the literature, the MS is still widely chosen as the dielectric boundary. The difference between this choice and the vdW surface is that, according to the latter protocol, the many crevices in the protein interior are treated as part of the solvent high dielectric. These crevices are not accessible to a spherical solvent used in defining the MS, and hence their being treated as part of the solvent dielectric is perceived as unrealistic or undesirable. However, this perception is open to question. Water molecules can access protein interiors, as demonstrated by many protein X-ray structures with water occupying interior positions, by the observation of positionally disordered water molecules in a hydrophobic cavity of interleukin 1β [26] ( Figure 4.3 ), and by molecular dynamics simulations [27] . In proteins like myoglobin and acetylcholinesterase (featuring a deeply buried active site connected to the exterior only through a narrow gorge), access by small molecules like water, made possible by the dynamics of the proteins, is essential for biological functions. We suggest that the vdW protocol provides a way to account for water access to protein interiors. Failure to account for this important property is perhaps the cause for overprediction of p K a shifts by the MS protocol (which is often “corrected” by increasing the protein dielectric constant to 20). In principle the vdW protocol can be mimicked by the MS protocol with appropriately reduced atomic radii. However, it has been found the precise amount of radius reduction varies from protein to protein and thus mimicking one protocol by the other appears to be a futile exercise [23] . We will come back to the debate between MS and vdW in Section 4.3 .

      Figure 4.3 . The presence of water molecules inside a hydrophobic cavity of interleukin 1β. The cavity is separated from the bulk solution according to the MS criterion but connected to the bulk solution according to the vdW criterion. When the three water molecules are moved from separate positions in the bulk solution to the configurations shown inside the cavity, the MS protocol predicts an increase of 0.9 kcal/mol in electrostatic free energy whereas the vdW protocol predicts a decrease of −2.2 kcal/mol.

      The generalized Born (GB) model has been developed as a fast substitute of the PB equation [28–31] . The GB model can be tailored to match PB results for electrostatic solvation energies obtained by either the MS or the vdW protocol. The errors of GB results in reproducing the PB counterparts are at least of the order of typical mutational effects on binding affinities. Therefore caution should be exercised when applying the GB model to calculate mutational effects.

      There is also progress in the opposite direction, i.e., toward more accurate modeling of electrostatic effects, by accounting for electronic polarization via quantum mechanical treatments [32,33] . Such treatments have not been used to directly predict the effects of mutations on the binding free energy, but it is already clear that electronic polarization can significantly influence electrostatic contributions to binding.

      Comparing PB or GB calculations against experimental data for mutational effects on binding affinity is premised on the assumption that the mutational effects are assumed to be dominated by electrostatic contributions. That is, possible contributions by hydrophobic interactions and by changes of conformational entropy are not taken into consideration.

      Watch the video: Πώς να βάλετε τη σωστή δόση σκόνης απορρυπαντικού-Ariel (January 2022).