Two M2 constructs, M2TM (residues 22–46) and M2(21–61) were synthesized using Fmoc solid-phase chemistry (PrimmBiotech, Cambridge, MA) and purified to >95% purity. Uniformly 13C, 15N-labeled amino acids (Sigma-Aldrich and Cambridge Isotope Laboratories) were incorporated at residues V27, S31, G34 and D44. The first three labeled residues test the pore-binding site, whereas the labeled D44 tests the presence of the surface binding site. Most other residues implicated in surface binding by the solution NMR study13 (link) showed longer distances to Rmt than D44, and thus were not labeled. Unlabeled peptides were used for static 2H quadrupolar echo experiments to determine the number of drugs bound to the channel and the effect of membrane composition on drug binding.
The M2 peptides were reconstituted into lipid membranes by detergent dialysis. For the 13C, 15N-labeled peptides, the peptide : lipid molar ratios were 1:8 for M2TM and 1:15 for M2(21–61), which corresponded to similar mass ratios of ~ 1 : 2. Three lipid membranes were used in this study: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer and two mixed membranes mimicking the virus envelope lipid composition to different extents. The virus-mimetic (VM) membrane is composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), egg SPM, which contains predominantly saturated palmitoyl chains, and cholesterol at a molar ratio of 21% : 21% : 28% : 30%. The modified virus-mimetic (VM+) membrane contains 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), SPM and cholesterol at a molar ratio of 25.6% : 25.6% : 25.6% : 23%. Thus, the cholesterol mole fraction is moderately reduced in the VM+ membrane.
For the mixed membrane samples, the lipids were codissolved in chloroform and methanol and then dried under a stream of nitrogen gas to remove the bulk of the organic solvents. The film was redissolved in cyclohexane, frozen and lyophilized to obtain a completely dry homogeneous powder. This lipid powder was suspended in a pH 7.5 phosphate buffer (10 mM Na2HPO4/NaH2PO4, 1 mM EDTA, 0.1 mM NaN3) and freeze-thawed 6 times to produce a uniform vesicle suspension. The peptides were reconstituted into the lipid vesicles by dialysis using octylglucoside.21 (link) The proteoliposome mixtures were centrifuged at 150,000 g to obtain ~40% hydrated membrane pellets, which were packed in 4 mm rotors for solid-state NMR experiments. Perdeuterated amantadine (d15-Amt) was directly titrated into the membrane pellet. After pellet formation and drug addition, samples for static 2H NMR experiments were lyophilized and rehydrated to ~40% with 2H-depleted water to ensure that d15-Amt was the only source of the 2H NMR signal. For 13C-2H REDOR experiments, d15-Amt was added at a ratio of 1 or 5 drugs per tetramer, corresponding to drug/lipid molar ratios of 1 : 60 or 1 : 12, respectively.
The M2 peptides were reconstituted into lipid membranes by detergent dialysis. For the 13C, 15N-labeled peptides, the peptide : lipid molar ratios were 1:8 for M2TM and 1:15 for M2(21–61), which corresponded to similar mass ratios of ~ 1 : 2. Three lipid membranes were used in this study: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer and two mixed membranes mimicking the virus envelope lipid composition to different extents. The virus-mimetic (VM) membrane is composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), egg SPM, which contains predominantly saturated palmitoyl chains, and cholesterol at a molar ratio of 21% : 21% : 28% : 30%. The modified virus-mimetic (VM+) membrane contains 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), SPM and cholesterol at a molar ratio of 25.6% : 25.6% : 25.6% : 23%. Thus, the cholesterol mole fraction is moderately reduced in the VM+ membrane.
For the mixed membrane samples, the lipids were codissolved in chloroform and methanol and then dried under a stream of nitrogen gas to remove the bulk of the organic solvents. The film was redissolved in cyclohexane, frozen and lyophilized to obtain a completely dry homogeneous powder. This lipid powder was suspended in a pH 7.5 phosphate buffer (10 mM Na2HPO4/NaH2PO4, 1 mM EDTA, 0.1 mM NaN3) and freeze-thawed 6 times to produce a uniform vesicle suspension. The peptides were reconstituted into the lipid vesicles by dialysis using octylglucoside.21 (link) The proteoliposome mixtures were centrifuged at 150,000 g to obtain ~40% hydrated membrane pellets, which were packed in 4 mm rotors for solid-state NMR experiments. Perdeuterated amantadine (d15-Amt) was directly titrated into the membrane pellet. After pellet formation and drug addition, samples for static 2H NMR experiments were lyophilized and rehydrated to ~40% with 2H-depleted water to ensure that d15-Amt was the only source of the 2H NMR signal. For 13C-2H REDOR experiments, d15-Amt was added at a ratio of 1 or 5 drugs per tetramer, corresponding to drug/lipid molar ratios of 1 : 60 or 1 : 12, respectively.
