High-power relaxation dispersion of ubiquitin (with SH3c titrated in) The high-power relaxation dispersion experiments were measured using the 15N based constant time E-CPMG experiment for quantifying kinetics of exchange processes in ubiquitin happening on a micro-to-millisecond time-scale. The measurements were conducted in Bruker Avance 600 MHz and 950 MHz spectrometers fitted with cryoprobe-TCI (Neo console running Topspin 4.x (Bruker Biospin corporation)) at 277 K. The constant time (CT) CPMG delay is divided into two equal halves, sandwiching the U-element that ensures the equal contribution of anti-phase and in-phase relaxation to R2,eff. In the E-CPMG experiment performed here, refocusing pulses are applied with (hard pulse) γB1/2π (7143 Hz and 7407 Hz for 15N in the 950 MHz and 600 MHz spectrometers respectively) fields (corresponding to 15N hard pulses) for all refocusing frequencies, thus reducing any off-resonance effects that can affect the measurement of R2,eff. The R2,eff values were measured at CPMG frequencies (νCPMG) of 66.7 Hz, 133 Hz, 267 Hz, 400 Hz, 533 Hz, 667 Hz, 1333 Hz, 2000 Hz, 2667Hz, 3333 Hz, 4000 Hz, 4667 Hz, 5333 Hz and 6000 Hz. The constant volume of 200 μL of NMR samples was put inside 3 mm tubes (Hilgenberg 299 GmbH) in 20 mM sodium phosphate buffer, pH 6.5, containing 100 mM NaCl, 10mM TCEP, 0.05% (w/v) sodium azide, and 10% D2O. In all experiments the ubiquitin (15N labeled) concentration was 1 mM. The SH3c (unlabeled) concentration was varied from 0, 0.02 mM, 0.05mM, 0.1 mM, 0.25 mM, 0.5 mM up to 1 mM. The probe temperature was calibrated using a digital thermometer and standard methanol sample. The reference spectra were collected without the CPMG delay period (τ). The R2,eff was calculated as R2,eff(νCPMG) = −1/T log(I(νCPMG)/I0) where νCPMG is the effective frequency of the CPMG field, (νCPMG = 1/(4τ), where the time between the centers of consecutive 180◦ pulses is 2τ), T is the constant delay during which CPMG pulses were applied (60 ms), I0 is the intensity of the peak in reference experiment (no constant-time delay) and I(ν) is the intensity of the peak at that particular CPMG frequency. The CPMG delay (60 ms) was chosen such that the residual intensity was approximately 50% of maximum intensity. The experiment was performed with 3 s recycle delay between increments using 12 different refocusing field strengths between 0 to 6000 Hz collected in scrambled and interleaved manner with 1024 (1H) and 130 (15N) complex points, respectively. For each increment, 16 transients were measured following the Echo-AntiEcho scheme for indirect frequency sign discrimination. There is a heat compensation block in the middle of the recycle delay that contains additional 180 degree pulses so that the total number of CPMG 180◦ refocusing pulses at fixed B1 field strength is constant irrespective of inter-pulse delay. The E-CPMG experiments took 3 days to complete, and standard 1H, 15N TROSY-HSQC spectra were collected before and after each experiment to monitor sample stability (there were no changes in the spectra before and after the experiments). The error-bars on individual data points reflect error-propagation of signal to noise ratio from duplicate measurement at one CPMG frequency. High-power relaxation dispersion of SH3c (with ubiquitin titrated in) The high-power relaxation dispersion on the 15N labeled SH3c were measured at 277 K in Bruker Avance-III 800 MHz spectrometer equipped with cryoprobe-TCI. The refocusing pulses were applied with γB1/2π ~5 kHz for 15N in an interleaved manner with 3 s recovery delay. The spectra were recorded with 1024 and 156 complex points in the direct and indirect dimensions, respectively. The NMR experiments were performed with the 15N-labeled CIN85-SH3 and unlabeled ubiquitin complex in 20 mM sodium phosphate buffer, pH 6.5, containing 100 mM NaCl, 10mM TCEP, 0.05% (w/v) sodium azide, and 10% D2O. In this experiment the SH3c (15N labeled) concentration was kept fixed at 1 mM, and the ubiquitin (unlabeled) concentration was varied from 0, 0.02 mM, 0.05 mM, 0.075 mM, 0.1 mM, 0.15 mM, 0.25 mM, 0.5 mM, up to 1mM. The R2,eff values were measured at the same frequencies as the previous experiment. In all other aspects, the experiment was performed under identical experimental setup as described above. Chromatograms and mass specs of 15N-Glu24-labelled G53(D)Thr ubiquitin are included.