Figure 6
Functional characterization of artificially Lys11-linked Ub2. (A,B) Close-up views of selected cross signals in two-dimensional 1H-15N HSQC spectra following the interaction of UBA2 from Rad23 with Lys11-linked Ub2 on the basis of isotopically labeled residues comprising the proximal moiety in this dimer. The increasing stoichiometry, n, of UBA2 regarding Lys11-linked Ub2 has been visualized by using a color coding ranging from red (n = 0), to orange (n = 0.3), to yellow (n = 0.9), to green (n = 1.7), and finally to blue (n = 3.1). The pronounced change in the chemical shift of I13 and T14 has been additionally highlighted by an arrow. (C) Overall change in chemical shifts of residues comprising the proximal moiety in Lys11-linked Ub2 comparing absence with presence of a three times excess of UBA2 regarding Ub2. The horizontal lines indicate Δω values larger than the mean (continuous mode) and larger than the mean plus one standard deviation (dotted mode). Residues undergoing a signal attenuation larger than 75% are highlighted by using a background colored in gray. Secondary structural elements according to PDB ID 1D3Z are indicated on top and the site used for conjugation has been highlighted by using a vertical arrow. Data for titrations of UBA2 to artificially Lys27-, and Lys63-linked Ub2s are shown in Fig. S16.
