Fig. 2: SEC-SAXS analyses of IM30* dimers.

a The average SAXS intensity (red dots) is compared to the normalized absorbance at 280 nm (black line) over the whole elution volume. b The scattering intensity after buffer subtraction was plotted against the scattering angle q. The red line represents the fit of the data for the pair distance distribution analysis by GNOM (χ² = 1.0392). c The pair distance distribution analysis in the range of q = 0.0929–7.2965 nm⁻¹ and forcing to 0 at D_max = 26 nm gave I₀ = 601.3 ± 4.5 cm⁻¹ and R_g = 6.86 ± 0.07 nm (total quality estimate from GNOM 0.59). d A dimensionless Kratky-plot was used to compare the scattering data obtained with IM30* and other proteins. Apparently, the Kratky curve of IM30 dimers lies in between the curves of the unfolded lysine riboswitch protein and the Plakin domain of Human plectin, which has an extended protein shape, clearly implying an extended and somewhat flexible structure of IM30* dimers. The dashed line indicates \(q \ast R_{\mathrm{g}} = \sqrt 3\). Black dots: Lysozyme (SASDA96)⁷². Red dots: Plakin domain of human plectin (SASDBC4)⁷³. Green dots: Unfolded lysine riboswitch (BIOISIS ID:2LYSRR)⁷⁴. Blue dots: IM30*. e The CD spectrum of IM30* (black squares) showed the typical characteristics of a mainly α-helical protein, i.e., pronounced minima at 222 and 208 nm. Yet, the amplitudes of the minima at 222 nm and 208 nearly doubled upon addition of 8 M TFE (red circles), which is known to induce α-helical structures in proteins/peptides. This implies that IM30* is highly unstructured. Error bars represent SD (n = 3). f The amplitudes of the minima at 222 nm and 208 nm of IM30 WT (black squares) only slightly increase upon addition of TFE (red circles), confirming the expected high content of α-helical structures. Error bars represent SD (n = 3). Based on the CD-spectra, the α-helix content of IM30* (e) was calculated to be ~57%, which is considerably lower than the reported and predicted α-helix content of IM30 WT of ~80%^16,17,18. In presence of TFE, both proteins reach about 100% α-helix content.