Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins

Abstract

To tackle the problems associated with membrane protein (MP) instability in detergent solutions,w ed esigned as eries of glycosyl-substituted dicarboxylate detergents (DCODs) in whichw eo ptimizedt he polar head to clamp the membrane domain by including,onone side,two carboxyl groups that form salt bridges with basic residues abundant at the membrane-cytoplasm interface of MPs and, on the other side,asugar to form hydrogen bonds.U pon extraction, the DCODs 8b, 8c,a nd 9b preserved the ATPase function of BmrA, an ATP-binding cassette pump,m uchm ore efficiently than reference or recently designed detergents.T he DCODs 8a, 8b, 8f, 9a,and 9b induced thermal shifts of 20 to 29 8 8Cfor BmrA and of 13 to 21 8 8Cf or the native version of the G-protein-coupled adenosine receptor A 2A R. Compounds 8f and 8g improved the diffraction resolution of BmrA crystals from 6t o4 .D CODs are therefore considered to be promising and powerful tools for the structural biology of MPs. Angewandte ChemieCommunications 2949

Stabilization of Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Using Novel Solubilization Agents

“…Due to its calixarene platform, calixarene detergent absorbs at 280 nm, which makes protein quantification difficult and limits the use of some biophysical charaterization, such as circular dichroism or tryptophan fluoresence. To adress this limitation, new classes of compounds with similar architecture but without the calixarene platform have been designed and applied successfully to membrane protein stabilization, 34 and this could be a promising future direction for MRP4 studies. The addition of mild groups such as saccharide heads or cholesterol-like groups provides more diversity for such classes of stabilizing detergents.…”

Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism

“…The elution peak was then pooled and stored at 4 °C before use. BmrA was particularly stable when not concentrated as previously reported (30). Thermostabilisation assays were carried out as previously reported (30).…”

“…Diffraction patterns of the resulting crystals displayed a lattice-doubling problem that prevented their processing and which we overcame by designing a series of tailored amphiphiles 3a-e (Fig. 1B; Chemistry section in Supplementary Materials) with a scaffold based on glycosyl-substituted dicarboxylates surfactants (30). Of note, these additives increase the thermal stability of BmrA up to ~30 °C for 3d (Fig.…”

“…BmrA was particularly stable when not concentrated as previously reported (30). Thermostabilisation assays were carried out as previously reported (30). Membranes of BmrA diluted at 2 g proteins/L were solubilized with 10 mM DDM, with or without 1 mM of compounds 3a-3e in a final volume of 2 mL, for 2 h at 4 °C.…”

Steroid‐Based Amphiphiles for Membrane Protein Study: The Importance of Alkyl Spacers for Protein Stability

“…Small amphipathic agents, in contrast, are widely used because they are not only efficient at extracting membrane proteins, but are also effective at preserving structural integrity during membrane protein purification and crystallisation. Representatives include hemifluorinated surfactants (HFSs), cholate‐ or resorcinarene‐based facial amphiphiles (FAs and RGAs), steroid‐based amphiphiles (e.g., chobimalt and GDN) and neopentyl glycol (NG) amphiphiles (GNGs and MNGs), pentasaccharide‐based amphiphiles (PSEs), mannitol‐based amphiphiles (MNAs), neopentyl glycol‐derived triglucosides (NDTs), dendronic trimaltosides (DTMs) and glycosyl‐substituted dicarboxylate detergents (DCODs) . Among these small amphipathic agents, steroid‐based amphiphiles such as chobimalt and GDN have received attention because cholesterol is an important component of eukaryotic membranes (Figure S1 in the Supporting Information), and using its derivatives [e.g., cholesteryl hemisuccinate (CHS)] as additives can improve protein stability through specific interaction with the membrane proteins .…”