Amphipols: polymers that keep membrane proteins soluble in aqueous solutions

Abstract

Amphipols are a new class of surfactants that make it possible to handle membrane proteins in detergent-free aqueous solution as though they were soluble proteins. The strongly hydrophilic backbone of these polymers is grafted with hydrophobic chains, making them amphiphilic. Amphipols are able to stabilize in aqueous solution under their native state four well-characterized integral membrane proteins: (i) bacteriorhodopsin, (ii) a bacterial photosynthetic reaction center, (iii) cytochrome b6f, and (iv) matrix porin.

Figure 1

Molecular structure of amphipols. The weight average apparent molecular weight (〈MW〉) was deduced from the 〈MW〉 of the polyacrylate precursors as estimated by gel permeation chromatography using narrow MW polyoxyethylene calibration standards (which may entail systematic errors). DP (〈MW〉/MW of monomer) is the corresponding number of units per chain. The number of monomers per chain in the most abundant molecules (representing ≈80% of the mass) actually ranges over one decade. x, y, and z are the molar percentages of each type of unit, randomly distributed along the chain. The average number of each type of unit per amphipol molecule is given between parentheses.

Figure 2

Solubility of membrane protein/amphipol complexes in aqueous solution. Aliquots from stock solutions of amphipols (5 g/liter in water) were added to purified membrane proteins in detergent solution and the mixtures diluted 10× with detergent-free buffer or with water. After 15 min incubation at 4°C, the solutions were centrifuged for 30 min at 4°C in the A-110 rotor of an Airfuge (Beckman) at 20 psi (1 psi = 6.89 kPa; ≈210,000 × g). The concentration of protein in the supernatant was determined from the absorbance at 564 nm (redox difference spectrum of b₆), 546 nm (BR), 278 nm (OmpF), or 802 nm (RC). (A) Cytochrome b₆f complex. Stock solution was ≈5 μM b₆f complex in 20 mM HG (cmc ≈19.5 mM), 0.1 g/liter EPC, 400 mM NaOH/AP buffer (pH 8.0). Final amphipol concentrations following 10× dilution with water were 0.5 g/liter (open bars) or 0.05 g/liter (solid bars). Control experiments included dilution with a 20 mM HG solution (Hecameg) or with water (buffer) in the absence of amphipols, and dilution with water in the presence of nonderivatized low MW polyacrylate (precursor). (B) Other proteins. Stock solutions: bacteriorhodopsin, ≈0.1 g/liter in 100 mM AP (pH 8.0), ≈10% sucrose, 10 mM OTG (cmc ≈9 mM); OmpF porin, ≈4 g/liter in 0.2% (wt/wt) octyl-POE (≈9.2 mM; cmc ≈7 mM) in the same buffer; reaction center, ≈3 g/liter in 20 mM HG in 20 mM NaOH·Tricine buffer (pH 8.0). Tenfold dilution with 100 mM AP (pH 8.0) to a final amphipol concentration of 0.5 g/liter.

Figure 3

Sedimentation velocity analysis of protein/amphipol complexes on detergent-free sucrose gradients. Aliquots (100 μl) of membrane proteins in detergent solution (cf. legend to Fig. 2) were supplemented with 1 g/liter A8-75 (plus, in the case of b₆f, 0.33 g/liter EPC), diluted 2-fold with 100 mM AP (pH 8.0), layered on top of 2-ml 5–20% (wt/wt) sucrose gradients in the same buffer and centrifuged at 54,000 rpm in the TLS55 rotor of a TL100 ultracentrifuge. After 5.25 h (RC), 5 h (b₆f), 6.5 h (OmpF), or 10 h (BR), gradients were collected by 120-μl fractions. Protein concentrations in the 16 fractions and in the resuspended pellet were determined spectrophotometrically (cf. legend to Fig. 2).

Figure 4

Stability of BR and cytochrome b₆f complexed by amphipols. (A) BR was either kept in 10 mM OTG in 100 mM AP, pH 8.0 (□; final concentration of OTG was 10 mM), or complexed by A8-75 (○) or A8-35 (•) as described in the legend of Fig. 2B (final concentration of amphipol was 0.5 g/liter). The three samples were stored at 4°C in the dark. Every second or third day, each sample was centrifuged in the Airfuge (cf. legend to Fig. 2) before measuring the absorbance at 546 nm. (B) Cytochrome b₆f was either kept in 20 mM HG, 0.1 g/liter EPC, 400 mM AP (pH 8.0) (▪), or complexed with A8-75 (▵) or A8-35 (▴) and isolated by sucrose gradient centrifugation as described in the legend of Fig. 3. The samples were stored at 4°C in the dark. Enzymatic activity was determined by diluting an aliquot 50- to 200-fold in 0.25 mM LM, 20 mM Tricine·NaOH buffer (pH 8.0), and measuring the rate of stigmatellin-sensitive electron transfer from decylplastoquinol to oxidized plastocyanin (14).

Figure 5

Sketch of an integral membrane protein complexed by amphipols. Protein and amphipols are drawn to scale (α-helix diameter, ≈1 nm; spacing of acrylic units along the polymer, ≈0.3 nm; length of octyl chains, ≈1 nm; expected mass of protein-bound amphipol, ≈20 kDa per mole of protein). The persistence length of the polymer, which determines the tightness of the loops, has been taken equal to ≈3 nm. It actually depends on such parameters as the density of charges along the chain and the ionic strength of the solution. The fractions of alkyl chains and polymer not in contact with the protein’s surface are largely speculative.