Fig. 6.

Subtle divergence between CRY1 and CRY2 at the secondary pocket largely dictates periodicity differences between the two repressors. a Surface view of the secondary pocket in CRY2 (PDB: 4I6E) with the seven divergent paralogous residues of CRY1 and CRY2 colored blue and labeled. CRY1 residues are shown in parentheses. b Rescue assays performed with WT CRY1, CRY1 3m (CRY1 M378V/K379R/E382D), and CRY1 5m (CRY1 E376S/M378V/K379R/E382D/W390F) shown as mean ± SEM (n = 5, 6, 6 per condition, reflective of 18 plates each from 6 independent experiments). c Period plot of rescues in b. Mean ± SEM indicated by bars. Asterisks show significance by unpaired t test with Welch’s correction (***p = 0.0005, ****p < 0.0001) compared to WT. d Rescue assays performed with WT CRY1, CRY1 7m (CRY1 G43A/N46S/E376S/M378V/K379R/E382D/W390F), WT CRY2, and CRY2 7m (CRY2 A61G/S64N/S394E/V396M/R397K/D400E/F408W) (n = 6 per condition, reflective of 30 (CRY1 7m), 33 (CRY2), and 24 (CRY2 7 m) plates from ≥8 independent experiments) shown as mean ± SEM. e Period plot of rescues in d. Mean ± SEM indicated by bars. Asterisks show significance by unpaired t test with Welch’s correction (*p = 0.0387, ****p < 0.0001). f Degradation assay with CRY1::LUC and CRY2::LUC and mutants (n = 6 per condition, 2 independent experiments). Samples were normalized to the initial luminescent signal. g Half-lives shown as mean ± SEM. Half-life was determined by fitting a one-phase decay curve to the data. Asterisks show significance by unpaired t test with Welch’s correction (*p < 0.05 (CRY2 vs. CRY1: p = 0.0407; CRY2 7 m vs. CRY1: p = 0.0376), NS (CRY1 7m vs. CRY1: p = 0.3484; CRY2 7m vs. CRY2: p = 0.9348; CRY2 vs. CRY1 7 m: p = 0.1483; CRY2 7m vs. CRY1 7 m: p = 0.1331)). h Correlation plot of period vs. half-life for the various pocket mutant rescues. Half-lives and periods shown as mean ± SEM. Linear regression is shown as a dotted line. The slope’s deviation from zero was not significant (p = 0.3418)