Avance 1 nmr spectrometer

Five millitres of mid-exponential phase bacterial cultures (grown in LB and M9 minimal salt medium at salinities ranging from 0.25 to 2.5 M NaCl) was harvested and centrifuged at 3000 g for 10 min. After the supernatant was discarded, the resulting pellets were washed twice in 1 ml sterile distilled water and vortexed for 1 min at room temperature. The washed cells were then transferred into 1.5 ml Eppendorf tubes and sonicated twice for 20 s with 10 s breaks between each 20 s of sonication to allow cooling of the sample. Samples were centrifuged at 6000 g for 10 min, and the resulting supernatant was transferred into 1.5 ml Eppendorf tubes, and stored at –80 °C. After a minimum of 2 h at −80 °C, the samples were freeze-dried and stored at room temperature until required for NMR analysis. The dried samples were prepared for NMR analysis by dissolving them in 500 µl of D₂O, adding 5 µl of 100 mM trimethyl silyl propionate (TSP; standard) using 1.5 ml Eppendorf tubes. The dissolved samples were transferred into 5 mm NMR tubes for analysis. Samples were run on a Bruker Avance-I NMR spectrometer operating at 500 MHz, at 298 K. All spectra used 90° pulses and long relaxation delays for accurate quantitation.

All ssNMR spectra were recorded on a 9.4 Tesla Bruker Avance I NMR spectrometer, operating at frequencies of 400 MHz (¹H) and 100 MHz (¹³C). Magic angle spinning (MAS) frequency was 10 kHz for all experiments.
Labelled peptides were packed into kevlar inserts (Bruker) which in turn fit into standard zirconia 4 mm rotors (Bruker).
¹³C and ¹⁵N cross polarization (¹³C CP) experiments: The standard CP sequence in the Bruker pulse program library was used with ¹H 90° pulse length 2.5 μs, contact time 2.5 ms; during the contact pulse a ramped pulse was applied on ¹H with average spin lock field of 70 kHz. During acquisition, SPINAL64 decoupling at field strength 100 kHz was applied on ¹H. Chemical shifts were referenced to external glycine (α polymorph), using the methylene signal at 43.1 ppm relative to TMS (¹³C).
Variable temperature experiment: using a Bruker Variable Temperature Unit, the temperature of the sample was varied from 242 K to 342 K in steps of 5 K. At each temperature, the sample was allowed to equilibrate for up to 10 minutes before a standard ¹³C CP-MAS spectrum was recorded.

All organics solvents and chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) and Thermo Scientific (Waltham, MA, USA). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhodamine-PE) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Poly(dimethylsiloxane)-nitrobenzoxadiazole (PDMS-NBD) and poly(dimethylsiloxane)-block-poly(ethylene oxide) (PDMS₃₆-b-PEO₂₃, M_n 4000 g.mol^-1) were synthesized as previously described [20 (link)]. This copolymer is known to spontaneously form polymersomes with a membrane thickness of 9.9 ± 1.6 nm. Cationic switchable lipid (CSL) was synthesized as previously described [16 (link)]. Sucrose, calcein and all other chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). ¹H Nuclear Magnetic Resonance spectra were acquired at 298 K on a Bruker Avance I NMR spectrometer operating at 400 MHz, using trimethylsilane (TMS) as the internal standard and deuterated chloroform as solvent. Data were treated with TopSpin 4.0.7 (Bruker BioSpin, Wissembourg, France).

Experiments were carried out on an 11.7 T NMR Bruker Avance I spectrometer (Bruker BioSpin) operating at 500.19 MHz for ¹H, and 132.3 MHz for ²³Na, using a 5 mm double resonance broadband probe. The test tubes with different samples under investigation (aqueous solutions with different NaCl concentrations, agarose gel, ex vivo tissues) were placed inside the spectrometer where the sample temperature could be controlled using gas flow and a temperature sensor providing a precise, stable and reliable temperature regulation. After each desired temperature was reached, a standard free induction decay (FID) pulse sequence was used with a 90° pulse. The duration of the pulse is 11 and 9 μs for ¹H and ²³Na, respectively, and 8 averages were used with TR = 15 s for ¹H, and 0.5 s for ²³Na, dwell time dw = 100 μs, spectral width sw = 5 kHz, 16,384 data points per spectrum. Complex FIDs were acquired in digital quadrature detection (DQD) mode, a simultaneous acquisition mode in Bruker systems resulting in $sw=\frac{1}{2\mathrm{dw}}$ . All experiments were performed with the following exact spectrometer reference frequencies: $f_0^{\text{H}}=500.2031765\text{MHz}$ , $f_0^{\text{Na}}=132.3120951\text{MHz}$ (fixed ratio $f_0^{\text{H}}/f_0^{\text{Na}}=3.7804796$ ).