Non-ionic detergents contain uncharged hydrophilic glucose-derived or polyoxyethylene head groups. This type of detergent is considered to be mild as the destabilization they cause is almost completely reversible. This capacity is explained by the fact that non-ionic detergents typically disrupt protein-lipid (and lipid-lipid) but not protein-protein interactions, thus maintaining (or better to phrase—not interfering with) the native state of a protein. Based on this, the primary application of non-ionic detergents is in the study of various biologically active forms of membrane proteins. However, the degree of destabilization correlates well with the length of the acyl chain of the detergent. In this regard, short (C7–C10) hydrocarbon chains (e.g., octylglucoside and nonylmaltoside) can often lead to deactivation of the protein, in contrast to their corresponding intermediate (C12–C14) chain-length derivatives (e.g., dodecylmaltoside) [1].
Fig. 1. The non-ionic detergents micelle.
Non-ionic detergents can be further classified into two types: polyoxyethylene (and related detergents), and glycosidic detergents.
Fig. 2. Structure of Triton X-100.
Fig. 3. Structure of n-dodecyl-β-D-maltoside.
Due to the mild and nondenaturing characteristics of non-ionic detergents, they are frequently used in studies of membrane proteins when compared to other applications. It is reported that a majority of the detergents used in the purification and structural determination of membrane proteins are non-ionic detergents. According to the report from Stetsenko et al., the non-ionic detergents account for a large part of the top 10 detergents used for the structural analysis of membrane proteins [2]. They are n-dodecyl-β-D-maltopyranoside (DDM), n-decyl-β-D-maltopyranoside (DM), n-octyl-β-D-glucopyranoside (OG), n-nonyl β-D-glucopyranoside (NG), polyoxyethylene 8 dodecyl ether (C12E8), n-undecyl-β-D-maltopyranoside (UM or UDM), lauryl maltose neopentyl glycol (LMNG or MNG-3), Triton X-100 and digitonin, respectively. And DDM, DM, and OG dominate, both for purification and crystallization. Looking at the results, one can confidently say that the non-ionic detergents rule the field of structural studies on membrane proteins. In addition, according to the result of statistics analysis, DDM and DM are reported as the detergents of choice for more than a half of structural studies on membrane proteins. With an addition of UDM, Cymal-5, Cymal-6, OG and NG, the total fraction of sugar-based detergents is around 75% of structural studies on membrane proteins.
Fig. 4. The bar representation (in %) of different detergents used for (A) membrane proteins purification and (B) crystallization (up to 31 December 2016).
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Shoja, Maryam, et al. Cell Biochemistry and Biophysics. 2021;79:87-92.
Studying changes in the structural and functional behavior of lipases in detergent environments is critical to the detergent industry, as most detergents affect both behaviors. This study aimed to investigate the effect of non-ionic detergents, specifically Tween 20 and 80, on the kinetic and structural behavior of Pseudomonas aeruginosa lipase as a model bacterial lipase.
Effects of Tween 20 and 80 on Lipase
The effects of Tween 20 and Tween 80 on lipase in the absence of detergent and in the presence of detergent were investigated. The data shows that in the presence of these detergents, Km decreases while Vmax increases. The effect of Tween 80 on enzyme kinetic parameters was greater than that of Tween 20. The Km value was 0.66 mg/ml in the control group and 0.52 and 0.45 mg/ml in the presence of Tween 20 and 80, respectively. The Vmax of lipase in the control group was 0.11 OD/min, and the Vmax of the Tween 20 and 80 groups were 0.14 and 0.15 OD/min, respectively.
Siposova, Katarina, et al. Colloids and Surfaces B: Biointerfaces. 2019;173:709-18.
Amyloid aggregation by insulin exhibits two distinct phases in the presence of submicellar concentrations of TX-100. The first stage is the inhibition stage where the molar ratio of protein to detergent is 1:0.1~1:1. At this stage, TX-100 inhibits the formation of insulin amyloid fibrils by up to 60%. As the detergent concentration increases, the molar ratio of protein to detergent is approximately 1:1~1:10, and a second "morphological" phase appears. Under this condition, the steady-state fluorescence intensity of insulin fibrils is significantly enhanced and the morphology changes significantly.
Interaction of Triton X-100 with Insulin
For insulin alone, a very small peak at 222 nm and 209 nm characteristic of α-helices was observed. Small changes in the CD spectrum of insulin were observed upon addition of TX-100 at concentrations up to 100 μM. However, with increasing detergent concentration ≥300 µM, both the shape and intensity of the spectra changed significantly, suggesting a breakdown in insulin's structural integrity.
Tidemand, Frederik G., et al. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2022;1864(6):183884.
Recently, cyclic and pressurized membrane scaffold proteins (MSPs) used to form nanodisks have been explored and found to have higher time stability at high temperatures. Using a standard recombination scheme using bile salts as a recombinant detergent, it was found that two linear structures formed multiple lipid protein species, and the addition of N-dodecyl-B-D maltoside (DDM) in the recombination resulted in the formation of single species of nanodiscs for these MSPs.
• DDM has a positive effect on the self-assembly of nanodisks
The non-ionic detergent DDM was added to the ingredients of the standard cholate adjuvant regimen because the combination of DDM with cholate has previously been shown to be compatible with nanodisc formation. Data show SEC purification of lsMSP1D1ΔH5 nanodiscs reconstituted from prepolymers containing cholate and varying concentrations of DDM, as well as samples prepared with DDM-solubilized POPC and cholate-free samples. The formation of nanodisks exhibits DDM dependence, in which undesired secondary peaks at low retention volumes taper off relative to the expected nanodisc peak. In the sample containing only DDM, a single peak was observed.
• DDM Increases the Binding of Proteorhodopsin to Nano-disk
Proteorhodopsin (PR) was used as a membrane model protein because the intrinsic absorption of this protein at 530 nm allows specific visualization of PR-loaded nanodiscs during SEC runs. Since PR is mainly composed of transmembrane helices, the success criterion is a monodisperse peak with the same retention volume as the empty nanodisk, i.e., approximately 11 ml to 14 ml, depending on the length of the MSP. For all sample pairs, it is clear that DDM promotes PR binding: with DDM, there is no peak in the void volume and the area is larger. Finally, samples prepared with cyclic MSPS and DDM showed the clearest peaks at the expected retention volumes.
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