Fig. 7.
Measuring EAM input parameters by NMR. (A) Representative NMR TROSY cross-peaks of apo R-subunit (CBD-A and CBD-B) and the reference cAMP-bound and C-bound R-subunits. (B) Chemical shift correlation plots of CBD-A and CBD-B for the apo sample, where the slope represents the fraction of ‘off’ states in each domain. The closed and open circles represent 1H and 15N chemical shifts, respectively. Figures are adapted from Akimoto M, McNicholl ET, Ramkissoon A, Moleschi K, Taylor SS, Melacini G. Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα. PLoS Biol. 2015;13:e1002305. (C) Similar to panel (A), but with the addition of the W260A:Rp2 TROSY spectrum. (D) Similar to panel (B), but for the W260A:Rp2 sample. (E) Similar to panel (C), but with the addition of the WT:Rp2 TROSY spectrum. (F) Based on the TROSY cross-peaks in panel (E), the intensities of the minor and major peaks are measured, allowing for the calculation of the open vs. closed population ratios. This population ratio is used to estimate the ΔGAB, as shown in panel (G). (H) Similar to panel (A) right, but with the addition of the C:R:Rp2 TROSY spectrum. (I) Similar to panel (D), but for CBD-B of the C:R:Rp2 complex. (J) The fraction of ‘off’ state of CBD-B in the C:R:Rp2 sample can be used to estimate the ratio of state-specific association constants of C-subunit for R-subunit (ρC). Figures were adapted from Byun JA, Akimoto M, VanSchouwen B, Lazarou TS, Taylor SS, Melacini G. Allosteric pluripotency as revealed by protein kinase A. Sci Adv 2020;6:eabb1250. Reprinted with permission from AAAS.