FIGURE 4.

Paramagnetic relaxation enhancement of ¹⁵N-¹H resonances of the FBP21-tandem-WW domain upon binding of a bivalent PRS ligand, which comprises an N-terminal TEMPO label. The figure shows the ratio of peak heights for NH backbone resonances of FBP21-tandem-WW WT (A), the binding deficient “W29A/W70A” mutant (B), the single domain mutants W70A (C) and W29A (D) all titrated with a 6-fold excess of TEMPO-labeled bivalent SmB. The obtained ratios of peak heights were calculated by comparing signals from measurements with active and inactive TEMPO-labeled peptides. The structure of the TEMPO-labeled peptide is shown as an inlet in panel B. Chemical shift changes of NH backbone resonances are shown as an inlet (dotted lines) for the single domain mutants (C and D).