In vitro α_IIb-β₃ TM heterodimerization. (A-C) Spectral region of TROSY-HSQC spectra showing Gly⁷⁰² and Gly⁷⁰⁸ of the ²H/¹³C/¹⁵N-labeled β₃ TM segment in the absence and presence of unlabeled α_IIb TM peptide. One set of signals corresponds to the monomeric signals (A). The signal intensity ratios of monomeric-to-heterodimeric signals, but not their positions, depend on the α_IIb and β₃ peptide concentrations, demonstrating slow exchange kinetics on the NMR timescale between the 2 species. As evidenced by the disappearance of the monomer signals at higher peptide-to-bicelle ratios (C), heterodimerization is predominant. (D) In the presence of mutant α_IIb(R995A) peptide, heterodimerization is weakened. The signal intensity ratio of heterodimer-to-monomer signals drops from 1.56 to 0.14 (C,D), and the heterodimeric signals are shifted compared with the interaction with wild-type α_IIb peptide. (E) The effects of charge reversal mutations in the membrane-proximal regions of α_IIb or β₃ on the TMD interaction were analyzed, as in Figure 1.