Figure 5. Changes in NMR parameters for K63-Ub2 upon E64RP mutation.
(A) Chemical shift differences of the distal unit upon mutation. Inset, residues with relatively large chemical shift differences (>0.01 ppm) are mapped to the surface of the distal unit (colored red). (B) Decreases in intra-molecular inter-subunit PREs upon mutation with an MTS paramagnetic probe conjugated at K48C site. Lines simply connect the data points. PRE values characteristic of C1 and C2 states are indicated with cyan and yellow strips, respectively. Error bars indicate the SD in PRE measurements.
Figure 5—figure supplement 1. Structural basis for the perturbation of K63-Ub2 conformational space upon E64RP mutation.
Representative (A) C1 and (B) C2 closed-state structures. Glu64 in the proximal unit opposes Arg72 and Arg74 in the distal unit, shown as sticks. Judging from the structures, the charge reversal mutation could have a larger impact on the stability of C1 closed state than on C2 closed state of K63-Ub2.
Figure 5—figure supplement 2. Overlay of 2D NMR spectra for wild type K63-Ub2 and E64RP mutant at 50 µM with distal unit 15N-labeled.
Residues with relatively large chemical shift differences (>0.01 ppm) are labeled.