Fig. 2.

NMR spectroscopy maps the PHR binding site on the CRY1 tail. (A) ¹H–¹⁵N HSQC spectrum of the isolated ¹⁵N-labeled CRY1 tail at 120 µM. (B) ¹³C–¹⁵N CON spectra of the ¹³C,¹⁵N-labeled CRY1 tail at 120 µM alone (black) or in the presence of 120 µM CRY1 PHR (red) (C), proline-specific region of the ¹³C–¹⁵N CON spectrum of the ¹³C,¹⁵N-labeled CRY1 tail ± CRY1 PHR (as in B). (D) Schematic of proline-containing peptide backbone, highlighting the correlations visualized by ¹H–¹⁵N HSQC (blue box) or ¹³C–¹⁵N CON (red box). (E and F) Relative intensity (E) and chemical shift perturbation (Δδ) (F) of the ¹³C,¹⁵N-labeled CRY1 tail upon addition of equimolar CRY1 PHR. Asterisk, unassigned or overlapping peaks excluded from the analysis. Exon 11 is represented by a gray box. In E, dashed horizontal line represents the mean intensity ratio (I_tail+PHR/I_{tail alone}), while the dotted lines represent SEM. Peaks are referred to by the number of the nitrogen of the ¹³C–¹⁵N peptide bond. (G–K) Zoom-in on ¹³C–¹⁵N CON spectra peaks (¹³C carbonyl of F513, M514, Y516, H541, and H548, respectively) that are severely broadened or perturbed upon addition of CRY1 PHR. Residues along a C–N bond point to their corresponding peaks with a cyan arrow, and severely broadened peaks with intensities below the base contour level are labeled with a red “x.” (L) NMR intensity ratios (I_tail+PHR/I_{tail alone}) grouped by exon (mean ratio per exon ± SD). (M) PONDR prediction of a disorder in the CRY1 tail with an ordered minimum in exon 11. (N) Random coil index (RCI) predicts lack of secondary structure (model-free parameter S² < 0.7) from NMR chemical shift assignments.