Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

Abstract

Membrane proteins can be reconstituted in polymer-encased nanodiscs for studies under near-physiological conditions and in the absence of detergents, but traditional styrene-maleic acid copolymers used for this purpose suffer severely from buffer incompatibilities. We have recently introduced zwitterionic styrene-maleic amide copolymers (zSMAs) to overcome this limitation. Here, we compared the extraction and reconstitution of membrane proteins into lipid nanodiscs by a series of zSMAs with different styrene:maleic amide molar ratios, chain sizes, and molecular weight distributions. These copolymers solubilize, stabilize, and support membrane proteins in nanodiscs with different efficiencies depending on both the structure of the copolymers and the membrane proteins.

Figure 1

Reaction designs used to prepare zSMAs via RAFT polymerization (A) and SMAs and zSMAs using Lipodisq P[St-ran-MA] copolymers as precursors (B).

Figure 2

Solubilization of recombinant proteins from E. coli crude membranes. Membranes were incubated in 500 mM NaCl and 50 mM Tris/HCl, with 10% glycerol, pH 7.5, and 1% (w/v) of copolymer, and the samples were incubated for 2 h at RT for HR or at 37 °C for MsbA. For the detergent experiments, DDM was used at 1.5% for HR and a mixture of 2% DDM and 0.04% sodium cholate was used for MsbA. (A) Solubilization of HR. Det (n = 21): detergent; SMA (n = 20): 2:1 SMA from Malvern; zSMA: our synthetic zwitterionic copolymers that include 1:1 zSMA (n = 21) and 2:1 zSMA (n = 26) derived from the RAFT P(St-ran-MA) with molecular weights of 6.7 and 6.4 kDa, respectively; M zSMA: zwitterionic copolymers synthesized from Malvern SMA precursors that include 1:1 M zSMA (n = 15) and 2:1 M zSMA (n = 23) derived from the Malvern P(St-ran-MA) with molecular weights of 4.6 and 5.0 kDa, respectively. The white and red colors indicate 1:1 and 2:1 St:MA, respectively. Det solubilization was significantly higher than that with all polymers (P < 0.001). *Denotes P < 0.001 vs other copolymers; ^† denotes P < 0.02 vs 2:1 zSMA and 2:1 M zSMA. (B) Solubilization of MsbA. Det: n = 21; SMA: n = 20; 1:1 zSMA: n = 21; 2:1 zSMA: n = 25; 1:1 M zSMA: n = 15; and 2:1 M zSMA: n = 23. Det solubilization was significantly lower than that with all polymers (P < 0.001). *Denotes P < 0.01 vs other copolymers, except for 1:1 M zSMA (NS); ^†Denotes P < 0.001 vs corresponding M zSMAs; ^‡Denotes P<0.001 vs 1:1 zSMA. Data are means ± SEM.

Figure 3

Effects of copolymer size on HR and MsbA solubilization. Solubilization conditions were those of the basic protocol, except that 1% or 2.5% of the 2:1 zSMA with three different chain sizes were used: the S, M, and L represent zSMA derived from RAFT P(St-ran-MA) with molecular weights of 3.1, 6.4 and 12.0 kDa, respectively. Data were normalized to the average of 1% zSMA with medium molecular weight (“M”). *Denotes P < 0.05 vs the corresponding 1% value. Data are means ± SEM (n = 3 per condition).

Figure 4

Solubilization of reconstituted HR and MsbA in liposomes. (A) HR and MsbA solubilization. HR and MsbA in crude E. coli membranes or purified HR and MsbA reconstituted in liposomes formed by E. coli lipids were solubilized under the conditions of the basic protocol (Fig. 2). The 1:1 and 2:1 zSMA were derived from the RAFT P(St-ran-MA) copolymers with molecular weights of 6.7 and 6.4 kDa, respectively, and the M zSMA was derived from the 2:1 Malvern P(St-ran-MA) copolymers with molecular weight of 5.0 kDa. *Denotes P < 0.05 vs the corresponding value for crude membranes. Data are means ± SEM (n = 4–7 per condition). (B) Typical examples illustrating the hydrodynamic diameter distributions of zSMALPs and M zSMALPs determined by dynamic light scattering. The 2:1 SMALPs were prepared using the SMA derived from the 2:1 Malvern P(St-ran-MA) copolymers with molecular weight of 5.0 kDa, and the zSMALPs and M zSMALPs were prepared using corresponding zSMAs and M zSMA as described in A. (C) Summary of the average hydrodynamic diameter of nanodiscs formed by solubilization of proteoliposomes containing purified HR or MsbA. Values for HR- and MsbA-loaded nanodiscs were not statistically different and were pooled. SMALPs (n = 5), 1:1 zSMALPs (n = 9), 2:1 zSMALPs (n = 9) and 2:1 M zSMALPs (n = 8). Data are means ± SEM; *Denotes P < 0.01 vs zSMALPs.

Figure 5

Thermal stability of solubilized MsbA. Purified MsbA was studied in detergent (Det) and zSMALPs (solubilized from proteoliposomes). For these experiments the samples were heated to 65 °C for 15 min and MsbA in the supernatant, after centrifugation at 100,000 g for 45 min, was quantified on Western blots probed with an anti-His antibody. The 1:1 and 2:1 zSMALPs were prepared using the corresponding zSMAs derived from RAFT P(St-ran-MA) copolymers with molecular weights of 6.7 and 6.4 kDa, respectively. *Denotes P < 0.001 vs both copolymers; ^†Denotes P < 0.002 vs 2:1 zSMA. Data are means ± SEM (n = 4 per condition).

Figure 6

Activity of HR and MsbA reconstituted in polymer-encased nanodiscs. (A) Cl⁻-induced HR spectral shift. Spectral shifts were elicited by increasing Cl⁻ concentration from zero (black trace) to 250 mM by addition of NaCl (red trace). Examples of HR in proteoliposomes and 2:1 zSMALPs are shown. The intensity was normalized to the corresponding maximal intensity in 250 mM NaCl. The zSMALPs were prepared using 2:1 zSMA derived from RAFT P(St-ran-MA) copolymers with molecular weight of 6.4 kDa. See Supplementary Information for examples in detergent, 2:1 SMALPs, 1:1 zSMALPs and 2:1 M zSMALPs. (B) Cl⁻-induced HR spectral shifts summary. For each experiment, the wavelength at the maximal intensity was recorded in Cl⁻-free solution and after NaCl addition, and the differences are presented as means ± SEM (n = 3–4 per condition). The SMALPs, zSMALPs and M zSMALPs are the same as those described in Fig. 4. *Denotes P < 0.001 vs all other conditions. (C) MsbA ATPase activity. The ATPase activity of purified MsbA in 100 mM NaCl and 20 mM Tris/HCl, with 15% glycerol and 0.2 mM TCEP, pH 7.5, and 0.065% DDM and 0.04% sodium cholate (Det), or after reconstitution in liposomes (Proteoliposomes) or copolymer nanodiscs formed by 2:1 SMA (2:1 SMALPs), 1:1 zSMA (1:1 zSMALPs), 2:1 zSMA (2:1 zSMALPs), or 2:1 M zSMA (2:1 M zSMALPs). The SMALPs, zSMALPs and M zSMALPs are the same as those described in Fig. 4. Data are means ± SEM (n = 8–10 per condition), and were normalized to the average in Det (2.3 ± 0.1 ATP/s). *Denotes P < 0.001 vs Det; the activity in 2:1 SMALPs and 2:1 M zSMALPs was not different from zero.