A class of mild surfactants that keep integral membrane proteins water-soluble for functional studies and crystallization

Abstract

Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside ('C12-b-M') as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b(6 )f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.

Fig. 1

Representatives of the three surfactant families with rigid ring systems tested in this work. 4-(4′-propoxyphenyl)-trans-cyclohexyl-α-D-maltoside (PoPC-a-M), (1); trans-4-(4′-propylphenyl)cyclohexyl-β-D-maltoside (PPC-b-M), (2); trans-4-(trans-4′-propylcyclohexyl)cyclohexyl-α-D-maltoside (PCC-a-M), (3).

Figure 2

Thermostability of β₁AR_34-324 in mild surfactants. The receptor was partially purified in C12-b-M by Ni²⁺-affinity chromatography with the surfactant exchange performed in the final column washes. The receptor was eluted in the rele vant surfactant and the thermostability was immediately assessed by heating for 30 minutes, quenching on ice and measuring the amount of functional receptor remaining using a ³H-DHA binding assay (Supplementary information): red squares, PCC-a-M, T₅₀ = 40° C; yellow tri angles, C12-b-M, T₅₀ = 30° C; green inverted triangles, BPC-b-M, T₅₀ = 25° C; blue dia monds, PPC-b-M, T₅₀ = 21° C. The results shown are from a single representative experiment performed in duplicate with error bars representing the s.e.m. Other experiments performed using the surfactants above, but not all simultaneously, gave similar apparent T₅₀-s.

Fig. 3

Thermostability of the human receptors of the Hedgehog pathway, hPtc and hSmo, in mild surfactants. Thermostability of purified hPtc and hSmo. Samples of hPtc or hSmo purified in C12-b-M, PCC-a-M, PPC-b-M or BPC-b-M were heated 30 minutes at increasing temperatures ranging from 4 to 70°C prior to injection on the reference flow cell (Fc1) and measure flow cell (Fc2) (coupled with N-SHh for hPtc and with CPN for hSmo experiments). Sensorgrams were recorded and the percentage of remaining native hSmo (a) and hPtc (b) was determined (SI). Red squares correspond to PCC-a-M; yellow triangles to C12-b-M; green inverted triangles to BPC-b-M and blue diamonds to PPC-b-M. The results shown are from a single representative experiment performed in duplicate with error bars representing the s.e.m.. Several other experiments were performed and gave similar apparent T₅₀-s.

Figure 4

Stability of cytochrome b₆f exposed to low (0.2 mM) or high (5 mM) concentrations of either C12-b-M or PCC-a-M. Top. Migration of the b₆ f complex in sucrose gradients (10-30% w/w) prepared in 20 mM Tris/HCl buffer, pH 8.0, containing either 0.2 or 5 mM of either C12-b-M or PCC-a-M. Freshly prepared b₆f (4.9 μM) in 0.2 mM C12-b-M (Stroebel et al., 2003) was layered on the top of the gradients and ultracentrifuged for 4 h at 4°C at 55,000 rpm (260.000 × g) in a Beckman TLS 55 rotor. Monomerization, as observed in the presence of 5 mM C12-b-M, correlates with the inactivation of the complex (see refs. (Breyton et al., 1997; Popot, 2010)). Bottom. Kinetics of inactivation of the b₆f complex in the presence of either 0.2 or 5 mM of either C12-b-M ( circles, diamonds) or PCC-a-M (squares, triangles). The dimer bands were collected from the gradients and their electron-transfer activity assayed as described (Pierre et al., 1995) immediately after collection (t=0) and after storage at 4°C in the dark for up to 10 daysthree weeks.

Figure 5

Crystal structure of the dimer of cytochrome b₆f with the localisation of the structured PCC-a-M molecules (8 per monomer) seen from the membrane in CPK representation. The stromal side is at the top.