Fig. 7. Incorporation of TMSiPhe in clathrin to monitor the conformation changes.

a Schematic diagram of interaction between the clathrin and arrestin during the receptor endocytosis. β-arr1 is recruited to the receptor when the receptor is activated by specific agonists. The arrestin mediate receptor internalization by interacting with clathrin and AP2. The clathrin terminal domain (TD) (heavy chain) interacted with the c-tail of the β-arr1. Residues of L82, K96, and K98 are close to the arrestin/clathrin interface (clathrin crystal structure, PDB: 1BPO). The TMSiPhe-incorporated clathrin sites are depicted as spheres with different colors (gray: L82, red: K96, blue: K98). The blue circles indicate the phosphorylation. b 1D ¹H NMR spectra of clathrin labeled as described in (7a). The spectra were recorded in a buffer containing 20 mm HEPES (pH = 7.5) 150 mm NaCl and 10% D₂O at 25 °C using a Bruker 950 MHz NMR spectrometer. The protein concentrations were 10 μm, and the total recording time per spectrum was 18 min. The chemical shift for the TMSiPhe protein was <−0.1 ppm. c–e 1D ¹H NMR spectra of clathrin alone and incubation with ISO-ppβ2V2R/β-arr1 complex. Clathrin-L82-TMSiPhe displayed no chemical shift in response to the incubation with the ppβ2V2R/β-arr1 complex. The clathrin-K96-TMSiPhe and clathrin-K98-TMSiPhe showed low-field shift (△shift = −0.005 ppm) and high-field shift (△shift = +0.004 ppm), respectively. The buffer used for the experiment contained 20 mm HEPES, 150 mm NaCl, 0.01% LMNG, 0.002% CHS, and 10% D₂O (pH = 7.5 at 25 °C). The spectra were recorded using a Bruker 950 MHz NMR spectrometer.