Fig. 3 : Characterization of the interaction of the IGFs with L-h IGFBP2. Sensorgrams of (a) IG F-1, (b) IGF-2 immobilized on the surface of a CM5 chip with L-h IGFBP2 as analyte. (c) Overlay of the 2D [15N-1H] HSQC spectra of L-h IGFBP2 (blue) and IGF-1: L-h IGFBP2 (red). Inset shows an expanded view of the region where residues show maximum chemical shift perturbations (residues 150-170). (d) Combined (15N and 1HN) chemical shift difference plot for L-h IGFBP2 residues upon addition of IGF-1, calculated using . The dotted line is shown at one standard deviation of the chemical shift differences. (e) Reported proteolysis sites on L-h IGFBP2. (f) Residues of the linker domain were analyzed for their predicted propensity to lie within MoRF motifs using the web-based program MoRFpred. A line between the peak centers of the red and blue signals for L152 and R156 residues shows the largest chemical shift deviations (in the insert, Fig. 3c ).
Fig. 4 : R2, R1ρ, and het-NOE plots for L-h IGFBP2 (blue) and IGF-1: L-h IGFBP2 (red) measured at 1H resonance frequency of 800 MHz. A-asterisks mark signals with overlap.
Fig. 5: Spectral density function values at J (0),J (ω N), andJ (0.87ω H) frequencies for L-h IGFBP2 (left) and the L-hIGFBP2:IGF-1 (right) complex at a 1H resonance frequency of 800 MHz. A-asterisks mark signals with overlap.
Fig. 6: The difference in S2 values (calculated using Eq. 1) between the free and bound forms of L-h IGFBP2 as estimated from J (0) andJN) calculated from R.
Fig. 7: Predicted structure of full-length IGFBP2 from AlphaFold protein structure database where the disordered linker domain is represented in red, starting from A97 residue to C191 as marked, with N- and C-terminal domains of FL-IGFBP2 represented in cyan and blue respectively. The helix in the linker domain was predicted by Alpha Fold only (https://alphafold.ebi.ac.uk/) and consistent with the fact that K150-E161 is more ordered in IGFBP2 as we can see from Fig. S4