For MAS solid-state NMR experiments, SGO_1180 dimer in the absence and presence of C-pep was reconstituted in 2H DMPC lipids. Specifically, recombinant 15N, 13C uniformly labeled SGO_1180 was solubilized in 0.1% DDM and added to a suspension of DMPC. The detergent was removed by addition of 50:1 (w/w) of biobeads. After centrifugation and removal of biobeads, the proteolipids were pelleted using two ultracentrifugation steps: the first one for 30 min at 30,000 rpm and a second at 80,000 rpm for 12 to 20 hours. Subsequently, the wet proteolipidic preparation was inserted in a 3.2-mm MAS rotor. For the preparation of the SGO_1180:C-pep complex, both proteins were solubilized in 0.1% DDM and added to the DMPC lipid vesicle preparation, reaching a final stoichiometry of 1:1 SGO_1180:C-pep (two C-pep per SGO_1180 dimer). A 50:1 (w/w) suspension of biobeads was used to remove DDM, and the complex was pelleted by ultracentrifugation before insertion in the MAS rotor. Solid-state NMR experiments were performed on an Agilent ssNMR spectrometer operating at a 1H Larmor frequency of 600 MHz equipped with 3.2-mm scroll MAS probe. For all the experiments, MAS rate (ωr) was set to 12 kHz, with acquisition time and recycle delay respectively set to 20 ms and 2 s. During acquisition, SPINAL-64 decoupling was applied (64). For cross-polarization (CP) and INEPT experiments, the temperature was respectively set to and 2° and 25°C (65). The 90° pulse lengths for 1H and 15N were 2.5 and 6 μs, respectively. The CP experiments were acquired with 32 scans, with the Hartman-Hahn contact time set to 500 μs during which the 15N radio frequency (RF) amplitude set to 35 kHz, whereas 1H RF amplitude was linearly ramped from 80 to 100% with the center of the slope set at 59 kHz (66). The 1D and 2D rINEPT spectra of SGO_1180 in the absence of SspA C-pep were acquired with 592 scans, whereas for the 1D rINEPT spectrum in the presence of SspA C-pep was acquired with 784 scans.

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