Ts. The phosphate transporter in the plasma membrane of Saccharomyces cerevisiae was effectively made in Pichia pastoris and purified in DPC detergent. Its activity was fully recovered after reconstitution in proteoliposomes with a similar substrate specificity as observed in an intact cell system.117 Conversely, opposite benefits were obtained with mitochondrial uncoupling proteins. The Chou laboratory reported protontransport activity for both UCP1 and UCP2 proteins in DPC,118,119 even though Zoonens and co-workers found that DPC fully inactivates both transporters.120 Asmar-Rovira and colleagues investigated how nine Chrysoobtusin Protocol detergents influence the function of the nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays.121 Below 45 mol of phospholipids per mole of nAChR, the receptor was rapidly inactivated. By very PD-161570 Epigenetic Reader Domain carefully measuring the amount of residual lipids soon after solubilization of enriched Torpedo membranes, they could show that most detergents degraded the receptor for the duration of purification below the critical threshold to sustain its activity. For example, Cymal-6, DDM, LDAO, and OG showed decreased stability and significant reduction or loss of ion-channel function. In contrast, CHAPS, DPC, and sodium cholate maintained stability and supported ion-channel function. Asmar-Rovira and colleagues concluded that within the case of nAChR, CHAPS, DPC, and sodium cholate mimic the lipids within the sense of being able to sustain lipiddependent activity and stability. The predicament is a lot more complex with all the human ABCG2 multidrug pump. MacDevitt et al. have been in a position to solubilize the recombinant protein from sf9 insect cell membranes only with hexadecyl phosphocholine.122 Immediately after three purification methods in hexadecyl phosphocholine, the protein was still in a position to bind the substrate, but its ATPase activity in detergent was low, and also the authors did not test ATPase activity after reconstitution in the protein in liposomes. They had been nonetheless in a position to analyze single particles by cryoEM and obtained a low-resolution threedimensional projection map displaying a tetrameric structure, which was interpreted as 4 homodimers of ABCG2. A second study appeared some years later, displaying that the ABCG2 receptor purified in hexadecyl phosphocholine was irreversibly inactivated, whilst exactly the same protein purified in DDM was active when reconstituted in liposomes containing an excess of cholesterol (40 ).123 The authors concluded that the homodimers of ABCG2 have been disrupted by hexadecyl phosphocholine, resulting within a comprehensive inactivation in the receptor.124 Similar final results have been obtained for BmrA, a multidrug resistance efflux pump. The protein was inactivated by DPC, but inside a reversible manner. Exchanging the alkyl phosphocholine detergent with DDM or anionic calix[4]arene-based detergents restored its activity. Reversible activation of pumps has also been noticed together with the human bile salt export pump, BSEP, produced in Pichia pastoris membranes and purified in phosphocholine detergents with linear or cyclic alkyl chains.125 Its activity was restored by exchanging the detergent with DDM.125 Inside the case on the multidrug resistance pump MDR3, addition of lipids towards the alkyl phosphocholine-MDR3 complex resulted inside a partial restoration of its activity.126 Apart from these examples of partial tolerance to DPC, you will find numerous examples of membrane proteins which are completely inactivated by this detergent (see Table S2). For example, diacylglycecol-kinase activity in the pres.
