Extended Data Fig. 8: NMR investigation of the N17 mutants exhibiting a different poly-Q helical propensity while preserving its length.

Uniformly labeled and SSIL samples of LKGG- and LLLF-H46 mutants were produced and analyzed by NMR. On one hand, LKGG-H46 glutamine ¹⁵N-HSQC signals (blue) collapsed in a broad, high-intensity, downfield-shifted peak, proving a substantial loss of helicity in comparison with the wild-type H46 (in gray) (a). Interestingly, this broad peak did not overlap with the positions corresponding to fully unstructured glutamines, which were shifted further downfield. This indicates that poly-Q, even when disconnected from the flanking region, contains a small intrinsic propensity for helical conformations, in agreement with our MD simulations. On the other hand, the ¹⁵N-HSQC spectrum of fully labeled LLLF-H46 (green) displayed a more dispersed density of glutamine peaks and an additional upfield density compared with the wild-type (in gray), pointing to a helicity increase of the poly-Q tract (b). The detailed analysis of NH, NεH₂ and Cα signals of Q18, Q20 and Q21 SSIL samples from both mutants confirmed the decrease in structuration of LKGG-H46 and the increase in helicity in LLLF-H46, in comparison with the wild-type form (in gray) (a,b and c). Importantly, Q21 in LLLF-H46 displayed signatures of two conformations as also observed for the wild-type. This residue exhibited two NHε correlation peaks, suggesting the formation of a stronger bifurcated hydrogen bond than in the wild-type. Unfortunately, a single Cα peak, corresponding to the less helical conformation, was observed for Q21 in this mutant, suggesting an unfavorable exchange regime for the NMR detection.