Ω76 synthesized by GenScript was further purified by reversed-phase HPLC using a Varian Pursuit XRs 5 C18 semi-preparatory column (bead size, 5 μm; pore size, 100 Å) connected to a Waters 1525 binary HPLC pump with a Waters 2489 dual wavelength ultraviolet-visible detector. A linear gradient of Milli-Q water and acetonitrile (HPLC grade, Merck), both with added 0.1% trifluoroacetic acid (Spectrochem), was used. The fraction containing Ω76 was subsequently concentrated by rotary evaporation and lyophilized to dryness. Samples were prepared for NMR experiments by dissolving lyophilized Ω76 in either 100% CD3OH (Sigma-Aldrich) or sterile Milli-Q water (10% D2O, Cambridge Isotope Laboratories) containing 25 mM dodecylphosphocholine-d38 (Isotec). A peptide concentration of 1 mM was used in all NMR experiments.

Data were acquired on a 600-MHz Agilent NMR spectrophotometer fitted with a triple resonance cryogenically cooled probe with a single (z axis) pulsed-field gradient accessory. Homonuclear 2D experiments—1H,1H-TOCSY (mixing time, 65 ms) and 1H,1H-NOESY (mixing time, 150 ms)—were acquired on both samples. All NMR data were processed and analyzed on an Apple Macintosh system running OS X 10.10. The spectra were processed and visualized using NMRPipe 9.6 (67), and further assignment and analysis was carried out using CcpNmr Analysis 2.4 (68).

Distance restraints were extracted from the intensities of the assigned peaks in the 1H,1H-NOESY spectra using the “Make Distance Restraints” tool in CcpNmr Analysis. The maximum upper distance limit used was 6.0 Å, and all lower distance restraints for proton-proton pairs were set to 1.8 Å. Secondary chemical shifts and characteristic sequential NOEs were used to assign secondary structure, and backbone dihedral restraints and backbone hydrogen bond restraints were set accordingly. CYANA 3.0 (69) was used to calculate 400 structures after 20,000 steps of simulated annealing in torsion angle space, and the 50 structures with the lowest target function were evaluated using the MolProbity (70) server. The first 30 structures with a clashscore of 0 were chosen for the final ensemble structure.

The structure ensemble was evaluated using the online PSVS (71) server. Root-mean-square deviation (RMSD) calculations were carried out in MOLMOL 1.0 (72). Further analyses were carried out on the first model. The electrostatic surface was calculated using the ABPS tool in UCSF (University of California, San Francisco) Chimera (73). The 3D-HM vector (61) was calculated using the online tool at www.ibg.kit.edu/HM/. The 3D-HM vector and electrostatic surface potential were calculated assuming a solvent dielectric constant of 20.00 for the micelle-water interface, as an interpolation between the polar solvent exterior and the nonpolar micelle interior.

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