AT3-UIM/Ub Interaction Figure 7. Isothermal Titration Calorimetry Experiments for the Interactions of various AT3-UIM12 forms with diUb types. A, B, C, Titration of different types of diUb with GB1-AT3-UIM12. A, Linear diUb; B, K48-linked diUb; C, K63-linked diUb. By fitting the data with the sequential binding model, these titrations are well-fitted to give two distinct binding affinities. D, E, F, Titration of K48-linked diUb with different AT3-UIM12 forms. D, GS-Sub; E, GS-Ins; F, RAP80-Linker. The concentration of K48-linked diUb is 100 mM and those of the AT3-UIM12 stocks are 2 mM. The titrations are well-fitted to a usual two-site binding model (dashed line) rather than the sequential binding model (solid line). The concentrations of diUb types are 100 mM and those of AT3-UIM12 stocks are 2 mM. The GB1 fusion was used for peptide expression and quantification. doi:10.1371/journal.pone.0013202.g007 minimal media using [15N]-NH4Cl and [13C]-glucose as the sole nitrogen and carbon sources (Cambridge Isotope Laboratories). After the GB1-fused AT3-UIM12 was eluted from Ni-NTA column, the GB1 tag was removed by thrombin digestion followed by a second Ni2+-NTA column and a Superdex-75 column. The resulting peptide fragments contain two extra residues, Gly and Ser, at the N-terminus and a His6 tag at the C-terminus. Purified peptides were dialyzed exhaustively against water, lyophilized, and stored at 220uC. Bacterial cells harboring pET-3a-Ub plasmid were grown at 37uC in LB or M9 minimal media until an OD600 of 0.6 was reached. Ub expression was induced at 22uC for about 10 h by adding IPTG to a final concentration of 0.2 mM. The protein was purified sequentially by acetic acid denaturation, 16/10 SP XL column and 16/60 Superdex-75 column chromatography. 15N- labeled Ub was prepared with 15NH4Cl as the sole nitrogen in M9 media. Purified Ub was dialyzed against water, lyophilized and stored at 220uC. The linear diUb protein was expressed and purified from bacterial cells harboring pET-22b-Ub2 plasmid. The K48- and K63-linked diUb proteins were prepared according to the literatures [52]. PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13202