Fig. 2. NMR characterizations of PD-L1-TC in bicelles.

(A) The 2D ¹H, ¹⁵N-TROSY-HSQC spectrum of PD-L1-TC samples in DMPC/DH⁶PC bicelles recorded at 310 K on a Bruker 600-MHz spectrometer. ppm, parts per million. (B) Cartoon representation of the 15 lowest-energy structures of PD-L1-TC in DMPC/DH⁶PC bicelles. The calculated helical region contains amino acids 238 to 261 (blue) with a length of 38.6 Å, covered by the predicted transmembrane region (amino acids 239 to 259, green) from UniProt (right). (C and D) Measurement of membrane immersion depth of PD-L1-TC in DMPC/DH⁶PC bicelles using the water-soluble probe Gd-DOTA. ¹H, ¹⁵N-TROSY NMR spectra of PD-L1-TC samples in the absence (red) and presence (black) of 15 mM Gd-DOTA; both spectra were acquired at 310 K on a Bruker 600-MHz spectrometer (C). PRE effects (I/I₀) versus [Gd-DOTA] for N236 and L248 were plotted at different concentrations of Gd-DOTA (D). I, peak intensity in the presence of Gd-DOTA; I₀, peak intensity in the absence of Gd-DOTA. (E) Assessment of PD-L1-TC in DMPC/DH⁶PC bicelles from the PRE effects of hydrophilic Gd-DOTA and lipophilic16-DSA paramagnetic probes. Peak intensity reductions induced by 16-DSA (2 mM) and Gd-DOTA (15 mM) relative to a reference spectrum are shown as bar graphs in orange and green, respectively. Measurements were carried out for PD-L1-TC in DMPC/DH⁶PC bicelles at an ¹H frequency of 700 MHz (pH 6.5) and 310 K. (F) Membrane partitioning of PD-L1-TC as determined by PRE effects. The position of the PD-L1-TC structure is shown along the membrane axis of the lipid bilayer. The transmembrane region (amino acids 239 to 261) is highlighted in gray, the juxtamembrane region (amino acids 262 to 272) is highlighted in light blue, and the cytoplasmic region (amino acids 273 to 290) is highlighted in pink, with residues 236, 251, and 261 colored green, red, and purple, respectively. The complete PREamp plot from Gd-DOTA titrations is shown on the right.