Designing Facial Amphiphiles for the Stabilization of Integral Membrane Proteins

Abstract

General procedure. NMR spectra were recorded on Bruker DRX-500, AMX-500 or AMX-300 instruments. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer. High-resolution mass spectra (HRMS) were recorded on a VG ZAB-ZSE mass spectrometer using MALDI (matrix-assisted laser-desorption ionization) or ESI (electrospray ionization).3α,7α,12α,24-Tetrahydroxycholane (3): [1] To a suspension of LiAlH 4 (3.85 g, 100 mmol) in dry THF (100 mL) at 0°C was added dropwise a solution of cholic acid (15.03 g, 35 mmol) in dry THF (200 mL) with vigorous stirring under nitrogen atmosphere. The reaction mixture was then heated to reflux with stirring for overnight. Upon completion, the reaction was carefully quenched with saturated aqueous NH 4 Cl solution at RT. Then the mixture was acidified with 1N HCl to pH 1~2. The precipitate was collected via filtration and washed with water and acetone to give the product 2 (12.1 g, 88%) as a white solid.7α,12α-Dihydroxycholane (2): To a solution of tetrahydroxycholane 3 (17.4 g, 44.2 mmol) and triethylamine (11.8 g, 116.6 mmol) in dry THF (300 mL) was added dropwise a solution of methanesulfonyl chloride (11.1 g, 97 .2 mmol) in dry THF (100 mL) at 0°C. Then the reaction mixture was slowly warmed up to RT. After 30 minutes, the reaction was quenched with saturated aqueous NH 4 Cl solution. The organic solvents were removed under vacuum and the left aqueous solution was extracted with ethyl acetate. The combined organic portions were washed with brine and then dried over anhydrous Na 2 SO 4 . The filtered solution was concentrated under vacuum to give the product 7α,12α-Dihydro-3α,24-dimethylsulfonate-cholane (25.6 g, 95%) which was directly dissolved in dry THF for the next step.A solution of LiAlH 4 (6.0 g, 158 mmol) in dry THF (300 mL) was added dropwise to the above obtained THF solution of 7α,12α-Dihydro-3α,24-dimethylsulfonate-cholane at 0°C. The reaction mixture was heated to reflux with stirring for overnight. Then the reaction was quenched with saturated aqueous NH 4 Cl solution at RT. The organic solvents were evaporated under vacuum and the left aqueous solution was acidified with 1N HCl to pH 1. The white precipitate formed was collected via filtration, washed with water and acetone to give the crude product, and then crystallized in dichloromethane and methanol to afford the pure compound 7α,12α-dihydrocholane (12.8 g, 80% over two steps 7α, 12α-Di-(O-β-D-maltosyl)-cholane (1):A mixture of compound 2 (210 mg, 0.58 mmol), 1-thio-ethyl-hepta-o-benzoyl-β-D-maltose (2.1 g, 2.08 mmol) and 4 Ǻ molecule sieves (800 mg) in dry CH 2 Cl 2 (50 mL) was stirred at RT for 30 minutes. The reaction mixture was then cooled to -15 °C, to which was added crystallized N-iodosuccinimide (500 mg, 2.22 mmol) and silver trifluorosulfonate (100 mg, 0.39 mmol). The reaction mixture was slowly warmed up to RT with stirring. The reaction was monitored by TLC. Upon completion, the reaction was quenched with triethylamine. The mixture was filtered and the filtrate was concentrated under vac...

Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins

“…We previously reported the synthesis of an FA (hereafter referred to as FA-1) with β-D-maltoside attached to the 7α-and 12α-positions in cholate (16). This unique FA design removed the flexible terminal polar head groups in cholate, CHAPS, and CHAPSO and attached polar side groups under the steroid surface.…”

An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization

“…Furthermore the steroid-based facial amphiphiles (FA) are another type of solubilization/stabilization agents being actively developed. They utilize the cholate scaffold, to which (typically) sugar headgroups are attached [82,83]. Such design proved to be useful to not only to stabilize membrane proteins (via tight interactions between a membrane proteins and FA owning to the large hydrophobic surface of the latter), but to also improve their crystallization (via the formation of more compact protein-detergent complexes, which can pack more tightly in a crystal, owing to the ability of FA to mediate intermolecular contacts between molecules of membrane proteins) [84].…”

Ice breaking in GPCR structural biology

“…McGregor et al have reported that lipopeptides (LPDs) are self-assembled into a cylindrical micelle with a width similar to that of a lipid bilayer, and further form a rigid sheath around the protein surface [55] . Zhang et al have developed cholate-based amphiphiles that project hydrophilic maltose units from one side of the steroidal skeleton, which is then further developed by the design of "tandem facial amphiphiles (TFAs)" [60,61] . The TFAs contain a pair of maltose-functionalized deoxycholate units, making it long enough to match the bilayer width.…”

“…These facial amphiphiles are very successful in reducing the micelle size. Both Zhang et al and Chae et al showed that the facial amphiphiles are only approximately 1/6 of the micelle size compared to the traditional detergents such as DDM, with an aggregation number of approximately 37 for FAs and only approximately 6 for the TFAs per PDC [60,61] . Although there is yet to be a direct application of these facial amphiphiles in Figure 3.…”

“…A brief summary of the new detergents to facilitate the GPCR structure study. (A) MNG [48] ; (B) branched detergent [47] ; (C) tandem facial amphiphiles (TFA) [61] ; (D) facial amphiphiles [60] ; (E) a cartoon about the working hypothesis of facial amphiphiles and tandem facial amphiphiles in compare with conventional detergents [60] .…”