5 doxyl stearic acid

Hexane, hexadecane, Na₂SO₄, MES, TRIS, EGTA, ammonium formiate, 5-doxyl stearic acid, DOPC, DOPE, cardiolipin and arachidonic acid were purchased from Sigma Aldrich GmbH (Germany). ONE, HNE and HHE came from Cayman Chemicals. E. coli polar lipid and DPhPC, were purchased from Avanti polar lipids. ULC/MS grade methanol was supplied by Biosolve BV (Valkenswaard, Netherlands). Chloroform was from Merck KGaA (Darmstadt, Germany).

The UCP2-FA samples were prepared using the UCP2-GDP NMR sample as a starting point. First, the buffer was exchanged using a G25 column equilibrated in Buffer C (30 mM potassium phosphate, 0.1% DPC, 0.05 mM GDP, and 80 mM NaCl, pH 6.5). The UCP2 containing fraction was concentrated to ~0.6 mM protein and then dialyzed against Buffer C using a 10 kD MW cutoff membrane. FA or ASO⁻ was added to the desired concentrations using a 100 mM stock solutions in 200 mM potassium phosphate (pH 6.5) and 2% DPC. For PRE experiments, we used a stock dispersion at 2.5 mM of FA-NO (5-DOXYL-stearic acid, Sigma) in 30 mM potassium phosphate (pH 6.5) and 1% DPC. Displacement of NO-FA by GDP was performed using a 0.6 mM UCP2 sample initially containing 0.3 mM NO-FA. GDP was added directly to the NMR sample as a stock solution (400 mM GDP, pH 6.5 and 0.1% DPC) to reach 0.1, 0.5, 1.0, 5.0 and 10 mM final GDP concentrations.
Since FA induced chemical shift changes are overall small, the backbone resonance assignments of the FA-bound UCP2 could be traced from those of GDP-bound UCP2 from earlier study (Berardi et al., 2011 (link)) by recording a series of 3D HNCO-TROSYs at different FA concentrations. The assignments of FA-bound UCP2 were also validated using a 3D HNCA-TROSY spectrum.