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. 2022 Jun 20;11:e76923. doi: 10.7554/eLife.76923

Figure 2. Myo-inositol has no effect on the native structure and only minor effects on stability.

(A, B) 1H-15N HSQC NMR spectra of full-length WT and W42Q HγD at 37 °C showed no evidence of interaction between myo-inositol and the protein’s native structure. (C, D) Differential scanning fluorometry with SYPRO Orange as the hydrophobicity probe revealed no change within the temperature range of aggregation but very small dose-dependent shifts toward higher melting temperatures. Normalized first derivative curves of SYPRO Orange fluorescence intensity are shown for both the N- and C-terminal domains, where peak positions correspond to Tm; insets show the difference curves. (E) Schematic of the design of the thermal scanning Raman spectroscopy apparatus. (F) Raman Amide I band spectra of the isolated N-terminal domain were likewise overlapping at low temperatures. (G, H) At 32.5 °C and 35.0 °C without inositol, a β-sheet peak ~1630 cm–1 became prominent, while a ~1620 cm–1 peak, typical of β-sheet-rich aggregates, was observed above 35.0 °C. Both features were strongly suppressed by 200 mM inositol.

Figure 2.

Figure 2—figure supplement 1. Histograms of chemical shift perturbations in WT and W42Q HγD upon addition of 100 mM myo-inositol.

Figure 2—figure supplement 1.

The NMR spectra are shown in Figure 2A and B. The total number of peaks was 152 for WT and 147 for W42Q. We used the Shapiro-Wilk test to determine whether the variability in chemical peak shifts beyond the minor systematic shift of ~0.008 ppm was randomly distributed. The largest chemical shift in either sample was 0.018 ppm (0.010 ppm from the mean). A normal distribution could not be ruled out for the CSPs of W42Q (p=0.13), while WT’s distribution had a marginal deviation from normality (p=0.011). Lack of a specific binding site is the most likely interpretation.
Figure 2—figure supplement 2. Secondary structure content of the WT and N terminal domain obtained upon deconvolution of the Amide I Raman spectra was comparable to the reported crystal structure (1HK0).

Figure 2—figure supplement 2.

(A) To check the quality of the Raman Spectra (Amide I range, 1600–1700 cm–1), we compared the secondary structure content (i.e. % of α-helix, β-sheet, and loops and turns) of the WT with the reported crystal structure (1HK0). Comparative bar plot shows comparable secondary structure content. (B) Comparative bar plot for the secondary structure contents shows N-terminal domain secondary structure content (at 25 °C) was comparable to the reported structure (1HK0-truncated N-terminal domain). Furthermore, the N-terminal domain with and without inositol had very similar secondary structure content, showing that inositol had no effect on the native secondary structure of the N-terminal domain (temperature = 25 °C).