The substitution of Ile232 to Thr232 induced substantial conformational changes in the TM domain of FcγRIIB as measured by solution NMR. (A and B) Overlay of ¹H-¹⁵N HSQC spectra (A) and 2D version of HNCO spectra of FcγRIIB-I232 and FcγRIIB-T232 (B). Spectra were recorded at 27°C and an ¹H frequency of 600 MHz using 0.1 mM ¹³C, ¹⁵N–labeled FcγRIIB-I232 or FcγRIIB-T232 reconstituted in a bicelle composed of 40 mM DHPC and 12 mM POPC in 20 mM Bis-Tris, pH 6.7, and 10% D₂O solution. The resonance signals of FcγRIIB-T232 were assigned by a set of triple resonance experiments and were labeled in the HSQC and HNCO spectras. ppm, parts per million. (C) The averaged chemical shift difference of the amide signals between FcγRIIB-I232 and FcγRIIB-T232 in the HSQC spectra. The polymorphism is shown by red dashed lines. The residues that had big chemical shift changes are shown by the dashed lines. (D) ¹H-¹³C HSQC was recorded at 27°C and an ¹H frequency of 900 MHz. The methyl signal (C^εH^ε₃) of the M222 sidechain is marked with a red asterisk. (E) PRE effects on the methyl signal of the M222 sidechain upon titration of Mn²⁺EDDA²⁻. The methyl signals of M222 in the presence of Mn²⁺EDDA²⁻ were normalized on the signal in the absence of the PRE reagent. (A–E) The results shown are one representative of at least two independent experiments. See also Fig. S2.