Ts. The phosphate transporter from the plasma membrane of Saccharomyces cerevisiae was successfully made in Pichia pastoris and purified in DPC detergent. Its 614726-85-1 Formula activity was completely recovered right after reconstitution in proteoliposomes using a similar substrate specificity as observed in an intact cell technique.117 Conversely, opposite outcomes have been obtained with mitochondrial uncoupling proteins. The Chou laboratory reported protontransport activity for both UCP1 and UCP2 proteins in DPC,118,119 while Zoonens and co-workers discovered that DPC totally inactivates both transporters.120 Asmar-Rovira and colleagues investigated how nine detergents influence the function on the nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays.121 Under 45 mol of phospholipids per mole of nAChR, the receptor was rapidly inactivated. By cautiously measuring the quantity of residual lipids soon after solubilization of enriched Torpedo membranes, they could show that most detergents degraded the receptor during purification under the vital threshold to sustain its activity. For instance, Cymal-6, DDM, LDAO, and OG showed decreased stability and substantial 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 inside the sense of becoming able to sustain lipiddependent activity and stability. The situation is even more complex with the human ABCG2 multidrug pump. MacDevitt et al. have been able to solubilize the recombinant protein from sf9 insect cell membranes only with hexadecyl phosphocholine.122 After three purification methods in hexadecyl phosphocholine, the protein was nevertheless able to bind the substrate, but its ATPase activity in detergent was low, and the authors did not test ATPase activity just after reconstitution in the protein in liposomes. They were nonetheless in a position to analyze single particles by cryoEM and obtained a low-resolution threedimensional projection map showing a tetrameric structure, which was interpreted as four homodimers of ABCG2. A second study appeared several years later, showing that the ABCG2 receptor purified in hexadecyl phosphocholine was irreversibly inactivated, while 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 had been disrupted by hexadecyl phosphocholine, resulting in a complete inactivation of your receptor.124 Related outcomes had been obtained for BmrA, a multidrug resistance efflux pump. The protein was inactivated by DPC, but within 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, created 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 from the multidrug resistance pump MDR3, addition of lipids towards the alkyl phosphocholine-MDR3 complex resulted in a partial restoration of its activity.126 Aside from these examples of partial tolerance to DPC, you’ll find many examples of membrane proteins which might be totally inactivated by this detergent (see Table S2). As an example, diacylglycecol-kinase activity inside the pres.
