5 dsa

The stock solution (2 mg/mL) of the spin label 5-doxyl stearic acid (5-DSA, Merck, Barueri, Brazil) was prepared in ethanol. Epimastigotes were spin-labeled as described by Alonso et al. [28 (link)] with minor modifications. Briefly, epimastigotes (5.0 × 10⁷ cells/mL) were incubated in 2 mL of LIT medium (without FBS) containing the compounds (5 × IC₅₀ and 10 × IC₅₀ values) for 24 h. Subsequently, the cells were washed with PBS (2500× g, 10 min), suspended in 50 µL of the same buffer, and each sample containing 1 x 10⁸ parasites was spin-labeled with 0.25 µL of 5-DSA ethanolic solution, and the system was gently mixed. The cells were then introduced into 1 mm capillary tubes, which were flame-sealed and centrifuged (2000× g, 5 min) to concentrate the parasites. EPR measurements were performed on the EPR EMX-Plus spectrometer (Bruker, Rheinstetten, Germany) using the following instrumental settings: microwave power, 2 mW; microwave frequency, 9.45 GHz; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; magnetic field scan, 100 G; sweep time, 168 s; and sample temperature, 25 ± 1 °C. The total scanning range of the magnetic field in each EPR spectrum was 100 G (x axis), and the intensity was measured in arbitrary units (y axis).

2D ¹H-¹⁵N-HSQC experiments of 0.5 mg of ^737-786gp36 CHR–MPER in DPC/SDS mixed micelles were acquired in the absence and in the presence of 5-doxylstearic acid (5-DSA). 5-DSA purchased from Merck was withdrawn from a 10 mM methanol-d₄ stock solution. All experiments were acquired with 512 increments of 256 time points and 32 scans on a 600 MHz Bruker Avance III spectrometer equipped with a TXI probe, running with TopSpin 2.1.

PoP-liposomes were formed with the inclusion 1% 5-DSA (Sigma, no. 253634) into the standard formulation using the thin film hydration method. A Bruker ER-200 ESR X-band spectrometer was used with a TE102 rectangular cavity and with a Bruker B-VT-1000 nitrogen flow temperature controller. The ESR frequency was 9.44 GHz, field modulation of 1.9 Gauss and microwave power of 0.64 mW. The sample size was ~20 μl in a 1 mm inner diameter quartz tube at the indicated temperatures. Samples were irradiated using a 658 nm 200 mW laser that was focused on the ESR sample through light slots on the Bruker X-band ESR cavity, which are conditions that induce PoP-liposome permeabilization.