Fig. 2: Methyl-TROSY NMR reveals conformational dynamics located around the N-terminal α1-helix.

a, Methyl-TROSY spectrum of U-²H, Ile-δ₁-labeled Xrn2 recorded at 800 MHz ¹H frequency and 313 K with assignments (Supplementary Methods and Supplementary Table 2). Isoleucines I467, I475, I504 and I544 are located in the flexible linker between CR1 and CR2 and were assigned by comparison of HMQC spectra from the Xrn2 WT protein and an Xrn2 ∆Linker construct. Isoleucines I748, I759 and I817 are in close spatial contact and lead to reciprocal chemical shift perturbations upon mutation; their isoleucine cluster (IC) was assigned to three peaks. The resonances of the I59, I89, I235 and I853 δ1-methyl groups are broadened. b, Distribution of isoleucine residues in the Xrn2 protein. Ile-δ1 probes are represented as spheres, where assigned probes are colored yellow, and unassigned probes are colored purple. Residues I59, I89, I235, I850 and I853 showed relaxation dispersion and are colored blue; their position is explicitly indicated. I59 and I89 are in close proximity and located above the central β-sheet opposite of I235. I850 and I853 are located in the C-terminal helix of Xrn2, with I853 directly opposite of Y14 at the rear side of the α1-helix. Methyl groups that could only be assigned at 293 K are shown in pink. c, MQ CPMG RD profiles measured at 313 K and 500 (yellow), 600 (red) and 800 (purple) MHz ¹H frequency. Data points are shown with error bars derived from multiple measurements; the curve corresponds to the best fit of the joined analysis of MQ CPMG and ¹³C-SQ CPMG data from all five residues. Fit values for | Δω_C | are given in the individual panels. Data points are shown as mean ± s.d., as derived from at least two duplicate NMR measurements.