New tensio‐active molecules stabilize a human G protein‐coupled receptor in solution

Abstract

Structural characterization of membrane proteins is hampered by the instability of the isolated proteins in detergent solutions. Here, we describe a new class of phospholipid-like surfactants that stabilize the G protein-coupled receptor, BLT1. These compounds, called C(13)U(9), C(13)U(19), C(15)U(25) and C(17)U(16), were synthesized by radical polymerization of Tris(hydroxymethyl) acrylamidomethane in the presence of thioglycerol, first endowed with two hydrocarbon chains with variable lengths (13-17 carbon atoms), as transfer reagent. C(13)U(19), C(17)U(16) or C(15)U(25) significantly enhanced the stability of BLT1 in solution compared to what was obtained with common detergents. These molecules therefore represent a promising step towards the structural characterization of BLT1 and possibly other membrane proteins.

Engineering a G protein‐coupled receptor for structural studies: Stabilization of the BLT1 receptor ground state

“…Mutations were confirmed by nucleotide sequencing. The unfolded wild‐type receptor and the different mutants were then expressed in Rosetta(DE3) E. coli strain inclusion bodies and refolded in fos‐choline‐16/asolectin mixed micelles in a buffer 12.5 m M Na‐borate, 100 m M NaCl, pH 7.8 as described in Damian et al 13…”

“…Buffer contributions were subtracted under the same experimental conditions. Circular dichroism (CD)‐monitored LTB 4 titration experiments were carried out as previously described 13. The spectra were the average of five scans using a bandwidth of 2 nm, a step‐width of 0.2 nm and a 0.5 s averaging time per point.…”

“…Different approaches have been developed so far to stabilize membrane proteins in solution. One possibility to alleviate protein unfolding by classical detergents is to use less aggressive surfactants 11–14. An alternative approach consists in mutating the protein to increase its stability in a wide range of conditions.…”

“…However, as stated above, an analysis of the structure of BLT1 requires the receptor to be stable in detergent solutions under conditions compatible with crystallization assays. We previously explored the role of surfactants on BLT1 stability 13. To achieve the highest stability of the purified BLT1 receptor in detergent solutions, we explored here the complementary approach that consists in producing a mutant of the receptor stabilized in its ground state.…”

Manipulation des récepteurs couplés aux protéines G

“…D'autres surfactants originaux ont été décrits, tels que des composés bicaténaires mimant la structure des phospholipides membranaires ou encore des composés de type lipopeptide. Ces molécules ont permis de maintenir certaines protéines membranaires en solution, mais l'extension aux RCPG reste encore anecdotique [36].…”