2.1 NMR Studies of L-hIGFBP2
L-h IGFBP2 was cloned, overexpressed, and purified as described in Materials and Methods. The purified protein had a molecular mass of 12.2 kDa as verified by MALDI-TOF mass spectrometry (expected: 12.211 kDa) and migrated at ~20 kDa on SDS-PAGE (Fig. S1 ). Such aberrant mobility on SDS-PAGE is typical of intrinsically disordered proteins (IDPs) and has been described for the linker domain of IGFBP5. The 2D [15N-1H] heteronuclear single quantum coherence (HSQC) spectrum of recombinant L-h IGFBP2 (Fig. 2a ) shows a unique15N-1H correlation for each residue, indicating that conformational averaging is fast on the NMR time scale. Comparison of the 2D [15N-1H] HSQC spectra (Fig. S2 ) for L-h IGFBP2 (in red) and full-length h IGFBP2(in blue) shows mainly overlap of all residues in the linker region regardless of whether they are part of the full-length protein or in isolation, implying that the linker domain is also disordered in the full-length protein.
The limited 1H chemical shift dispersion of the 2D [15N-1H] HSQC spectrum implies that L-h IGFBP2 is unstructured. In support of this,3J HNHα coupling constants (measured using G-matrix Fourier transform (GFT) (3,2) D HNHA), a lack of deviations of backbone chemical shifts from random coil values, and secondary structure propensity prediction also indicate an overall lack of ordered secondary structure (Fig. S3 ). The NH bond order parameter estimation based on the chemical shifts of the backbone (1Hα,13Cα, 13C′,15N) and sidechain (13Cβ) nuclei confirms a high degree of disorder in L-h IGFBP2 (Fig. 2b ), in agreement with the general consensus that the linker domains of all IGFBPs are disordered (Fig. S4 ). The backbone1H amide exchange rates could not be obtained from 2D15N-1H HSQC spectra, as the decrease in intensity of cross-peaks in the 2D spectrum upon dissolving the protein in 100 % 2H2O was too fast to be measurable. The exchange rates were therefore characterized from 2D13CO-15N EXSY at pH 6 and 20 °C, which indicated an upper limit of kex~1 s-1 for k exfor all residues for a 1:1 mixture of H2O and2H2O (Fig. S5).