Table 1. Apparent Dissociation Constants for the Ub Binding Affinities of Tandem AT3-UIM12, Single AT3-UIM Motifs and Their Mutants.
| UIM Peptide | K D,app (µM)a |
| AT3-UIM12 (WT) | 97.0±16.9b |
| AT3-UIM1 | 215±32 |
| AT3-UIM2 | 309±30 |
| AT3-UIM3 | > 3000 |
| AT3-UIM12-I240A | 141±9 |
| AT3-UIM12-I240P | 175±3 |
| AT3-UIM12-Q238E/E239T | 333±23 |
| GS-Subc | 195±17 |
| GS-Ins | 16.8±8.3 |
| RAP80-Linker | 39.0±11.5 |
a
The dissociation constants were determined from NMR titration of different GB1-tagged UIM peptides to 15N-labeled Ub. For comparison, we calculated the apparent K D,app values for AT3-UIM12, provided that the two UIM motifs bind Ub equally.
b
The chemical shift changes of three individual residues I13, K48 and L71 on Ub were used to calculate the dissociation constants. Data are presented as mean ± SD.
c
The sequences of the linker regions for wild-type AT3-UIM12 and its mutants. WT: 239EIDMED244; GS-Sub: GSSGGS; GS-Ins: EIDGSSGGSMED; RAP80-Linker: EAREVNNQEEK.