Mouse UCP2 (residues 14–309, with a C-terminal His6 tag) was expressed using a pET-21 vector in E. Coli Rossetta DE3 cells. After cell lysis, the lipid composition of the membrane fraction was adjusted by adding DMPC, cardiolipin, and phytanoyl lipids. The protein was extracted using 0.2% DPC in the presence of GDP. The solubilized UCP2 was then subjected to a series of purification steps including Ni-NTA affinity, MonoQ ion exchange, nucleotide-analog affinity, and size exclusion chromatography. The final NMR sample contained 0.8 mM UCP2, 5 mM GDP, 150 mM DPC, 2 mM DMPC, 1 mM cardiolipin, 5 mM β-mercatpoethanol, 30 mM potassium phosphate (pH 6.5), and 80 mM NaCl.
NMR experiments were conducted at 33 °C on spectrometers equipped with cryogenic probes. Sequence specific assignment of backbone chemical shifts was accomplished using three pairs of triple-resonance experiments and a double 15N-edited NOESY, recorded on (15N-, 13C, 2H) labeled protein. RDCs were obtained using an aligned sample containing 0.5 mM UCP2 and 20 mg/ml DNA nanotube19 (link) (other components same as above). 1DNH was measured using the J-scaled TROSY-HNCO experiment. 1DC’Cα and 1DNC’ were measured using TROSY-HNCO with quantitative-JC’Cα and -JNC’ modulations, respectively. For obtaining PREs, we generated a Cys-less UCP2 mutant and introduced single cysteines at desired positions for labeling with MTSL (METHODS). Residue-specific broadening of protein resonances was measured with two TROSY-HNCO spectra, one recorded after nitroxide labeling and another after reducing the nitroxide free electron with ascorbic acid.
Structure determination had two stages: 1) determining local structural segments by RDC-based MFR protocol and 2) determining the spatial arrangement of the MFR-derived segments using PRE distance restraints. Structures were calculated using XPLOR-NIH30 with backbone ϕand ψ of the assigned structured segments, RDCs, and PRE-derived distances. A total of 30 structures were calculated using a simulated annealing protocol, and 15 low-energy structures were selected as the structural ensemble (statistics inSupplementary Table 1 ).
NMR experiments were conducted at 33 °C on spectrometers equipped with cryogenic probes. Sequence specific assignment of backbone chemical shifts was accomplished using three pairs of triple-resonance experiments and a double 15N-edited NOESY, recorded on (15N-, 13C, 2H) labeled protein. RDCs were obtained using an aligned sample containing 0.5 mM UCP2 and 20 mg/ml DNA nanotube19 (link) (other components same as above). 1DNH was measured using the J-scaled TROSY-HNCO experiment. 1DC’Cα and 1DNC’ were measured using TROSY-HNCO with quantitative-JC’Cα and -JNC’ modulations, respectively. For obtaining PREs, we generated a Cys-less UCP2 mutant and introduced single cysteines at desired positions for labeling with MTSL (METHODS). Residue-specific broadening of protein resonances was measured with two TROSY-HNCO spectra, one recorded after nitroxide labeling and another after reducing the nitroxide free electron with ascorbic acid.
Structure determination had two stages: 1) determining local structural segments by RDC-based MFR protocol and 2) determining the spatial arrangement of the MFR-derived segments using PRE distance restraints. Structures were calculated using XPLOR-NIH30 with backbone ϕand ψ of the assigned structured segments, RDCs, and PRE-derived distances. A total of 30 structures were calculated using a simulated annealing protocol, and 15 low-energy structures were selected as the structural ensemble (statistics in
