Amphipols: Amphipathic polymers that stabilize membrane proteins in aqueous solutions

Rationale. In a solution of detergent-solubilized membrane protein (MP), detergent molecules are in a constant equilibrium between monomers, micelles, and the surfactant layer that covers the transmembrane region of the protein and makes it hydrophilic. To prevent MPs from aggregating, they must be handled above the cmc of the detergent, i.e. in the presence of free micelles. The latter act as a sink for hydrophobic and amphipathic molecules, e.g. lipids, which is a major cause of MP inactivation. This could in principle be alleviated by designing molecules with such a high affinity for the transmembrane surface of MPs that they would never dissociate. Thus, traces of free surfactant would suffice to keep the protein soluble. This concept led to the creation of 'amphipols' (APols), namely short amphipathic polymers that are able to keep individual MPs soluble under the form of small hydrophilic complexes (Fig. 1)

Figure 1. Chemical structure of amphipol A8-35; x ≈ 0.35, y ≈ 0.25, and z ≈ 0.4. The average number of acrylate units is ~70 and the average molecular mass 9-10 kDa. (From Tribet et al., PNAS, 1996)

Proof of concept. APols can be grafted with functional groups such as the fluorescent probe NBD, yielding a fluorescent APol (FAPol), without affecting their physical-chemical properties. FRET experiments show that extensive dilution of MP/FAPol particles into an APol-free medium does not entail any detectable desorption of FAPols, even after extended periods of time (hours-days) (Fig. 2, left). On the other hand, MP-bound FAPol readily exchanges for other surfactants, be they detergent or unlabelled APol (Fig. 2, right).

Figure 2. Left panel: Stability of the fluorescence of tOmpA/FAPol complexes after 1000x dilution into APol-free buffer, indicating the absence of dissociation. Right panel: Kinetics of exchange of tOmpA-bound FAPol for unlabelled APol after supplementation of a tOmpA/FAPol sample with a 50X excess of unlabelled APol: provided the repulsive electrostatic interactions between MP-bound and free APols are screened, the exchange is quite rapid (minutes). (From Zoonens et al., Biochemistry, 2007)

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