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. Author manuscript; available in PMC: 2011 Nov 26.
Published in final edited form as: J Mol Biol. 2010 Sep 25;404(2):274–290. doi: 10.1016/j.jmb.2010.09.044

Figure 4.

Figure 4

Analysis of the chemical shift changes in 15N-AP180 M5 upon titration with unlabeled clathrin TD. A,E: HSQC spectra of two representative amino acid residues within clathrin binding site 1 (A) and clathrin binding site 2 (E). As the [clathrin TD]total/[AP180 M5]total ratio increased, the peak positions shifted as indicated by progression from black to red to green to magenta colored peaks. The asterisks below the sequences indicate the residues used for the subsequent KD determination. Peaks that broadened extensively were omitted from the KD analysis. B,C. Determination of the KD of clathrin binding site 1 in the WT AP180 M5 (B), and in a single-site AP180 M5 in which binding site 2 was mutated (C). F,G: Determination of the KD of clathrin binding site 2 in WT AP180 M5 (F), and in a single-site AP180 M5 in which binding site 1 was mutated (G). Plotted (B,C,F,G) are the weighted average chemical shift changes of the 1H and 15N resonance of the amino acid residues. The data shown in each panel was globally fit to a hyperbolic equation, and fits are indicated with red traces. D,H: Determination of the dissociation rate constant koff of clathrin binding site 1 (D) and site 2 (H) in the WT AP180 M5. The line width analysis was performed as described in materials and methods, and global fits are indicated with red traces.