Figure 4.

(A) Overview of the procedure to obtain Integrin α_IIb [trans-membrane and cytoplasmic domains (TMCD)] incorporated nanodiscs. TEV protease treatment of MBP- Integrin α_IIb (TMCD) incorporated nanodiscs, cleaves the MBP–tag at the TEV cleavage site engineered between the tag and Integrin. The α_IIb incorporated discs can then be purified from the rest of the cut products using SEC. Inset: Negatively stained TEM image of α_IIb containing nanodiscs displaying a homogeneous population of discs. The exact same procedure can also be extended to obtain β₃ (TMCD) incorporated nanodiscs. (B) ¹H-¹⁵N- TROSY HSQC spectra of Integrin α_IIb and β₃ (TMCD) acquired on 800 MHz magnet (Agilent, USA). The sharp peaks correspond to the cytoplasmic tail region while the broader peaks correspond to the transmembrane region. (C) Applicability of nanodisc systems for studying signaling pathways. A schematic showing the activation events of Integrin β₃ which is relayed intracellularly through the phosphorylation of its cytoplasmic tail by Src kinase (Top Panel). This event is mimicked (in vitro) in an NMR tube by mixing ¹⁵N labeled β₃ incorporated discs with the kinase domain of Src kinase in the presence of ATP molecules. Phosphorylation is manifested through chemical shift perturbations (CSP) observed through the overlay of the ¹H-¹⁵N- TROSY HSQC spectra obtained from the un-phosphorylated (black) and bi-phosphorylated (red) β₃ (TMCD) collected on 600 MHz magnet (Agilent, USA). Also shown is the sequence of the β₃ tail where the phosphorylation occurs at the two tyrosine residues.