Figure 4. Slow isomerization of the TAD switch occurs on the timescale of minutes.

(A) Highlighted region of the 2D spectrum from a 15N-1H ZZ-exchange assay performed at 25°C of 15N BMAL1 TAD displaying cis (dark green), trans (dark blue), cis to trans (light green) and trans to cis (light blue) cross peaks for L626 at a delay = 1 s. Dashed circles, location of exchange cross peaks. (B) Overlay of 15N-1H HSQC spectra showing the cis and trans peaks of L626 at increasing temperatures. (C) Snapshot of ZZ-exchange assays at delay = 1 s at indicated temperatures. (D) Eyring analysis of exchange rates vs. temperature from ZZ-exchange assays with errors displayed in s.d. (E) Free energy plot showing the calculated activation energy of isomerization for the BMAL1 TAD. See also Figure S4.