Figure 4

¹H-¹⁵N HSQC spectra of Lt2 and Lt4 in the presence and absence of RRM1/2/3/4-poly(A). (a) Fragments of the linker-PABC region used in this study. (b,c) ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled Lt2 (b) and Lt4 (c) at 64.4 μM, in the presence (red) and absence (black) of 43.6 μM RRM1/2/3/4-poly(A). The peak labels in the parentheses in panel (b) represent the minor peaks of the split signals. The 9 NMR signals with peak labels enclosed by the red box in the panel (b) did not disappear upon addition of RRM1/2/3/4-poly(A). (d) ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled Lt2 at 100 μM in the presence and absence of RRM1/2/3/4-A₂₄ at 50 (blue), 100 (green) and 150 μM (red). The NMR signal of W437 in the black box is enlarged. (e) The estimation of the dissociation constant (K_d). The chemical shift changes of W437 sidechain (sc), W437 and T438 were plotted against RRM1/2/3/4-A₂₄ concentration in ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled Lt2 at 100 μM in the presence and absence of RRM1/2/3/4-A₂₄ at 50, 100 and 150 μM. The K_d values were estimated by the curve fitting of the chemical shift changes of the NMR signals for W437sc, W437 and T438, by a 1:1 binding model using the formula,\({\rm{\Delta }}{\rm{\delta }}={\rm{\Delta }}{\rm{\delta }}\,\max \,\times \frac{({\rm{X}}+[{\rm{Lt}}2]+{\rm{Kd}})-\sqrt{{({\rm{X}}+[{\rm{Lt}}2]+{\rm{Kd}})}^{2}-4{\rm{X}}[{\rm{Lt}}2]}}{2[{\rm{Lt}}2]}\)(4) where Δδ, Δδ max, X, and [Lt2] are the chemical shift changes, maximum of the chemical shift changes, and the concentrations of RRM1/2/3/4-A₂₄, and Lt2 (100 μM), respectively. Curve fitting was performed using Origin 5.0 software. (f) The chemical shift changes between 0 and 150 μM RRM1/2/3/4-A₂₄ are plotted versus the residues of Lt2. The chemical shift changes were calculated using the equation (3) in the Methods. *Indicates Pro or residues without assignments.