Fig. 5: CEST and stopped-flow spectroscopy analyses of DREB2A showing two bound states.

A Example ¹⁵N-CEST profiles for DREB2A Gly243 in the trans and cis proline states, illustrating binding of both isomers, and Phe274 illustrating three-state behavior of sequentially distant residues. Shown profiles were recorded with (colored) and without (black) 5% unlabeled Med25-ACID at 25 °C. Vertical gray lines indicate the chemical shifts of detected populations, with the solid line marking the free state and dashed line(s) marking the bound state(s). B Kinetic model for analysis of the ¹⁵N-CEST data with derived affinities. Exchange between the cis and trans bound states is not considered. C DREB2A ¹⁵N CSPs extracted from the CEST experiment. The dashed line indicates an arbitrary significance level of 2.2 ppm. D Mapping of DREB2A ¹⁵N CSP on the AF2 complex model. Residues with significant CSPs are shown. E Stopped-flow fluorescence spectroscopy analysis of ABS (top) and ABS-RIM (bottom) to determine association (left) and dissociation (right) rate constants. Data points and error bars represent mean and standard deviation of 19 or more individually fitted traces assuming a normal distribution. Example averaged traces are shown for each analysis. Averaged traces and derived k_obs for all data points are shown in Supplementary Figs. 11 and 12. Source data are provided as a Source Data file.