Extended Data Fig. 2. Biochemical and NMR analysis of isolated aromatic variants.

(a) Aromatic residues that have been mutated in this study mapped onto the PDB structure of FLN5 (PDB 1QFH). (b) POPMUSIC stability predictions of the FLN5 aromatic mutations⁶⁵. (c) Western blot (anti-His tag) of an expression text of FLN5, FLN5 Y719E, FLN5 A₃Y₃, FLN5 F₃A₃ and FLN5 A₃A₃ proteins (L: total lysate, S: soluble lysate) and Coomassie stained SDS PAGE gel of purified FLN5 Y719E, FLN5 A₃Y₃, FLN5 F₃A₃ and FLN5 A₃A₃ proteins. (d–f) ¹H,¹⁵N correlation spectrum (283 K) of FLN5 Y719E overlaid with that of (d) FLN5 A₃Y₃, (e) FLN5 F₃A₃ and (f) FLN5 A₃A₃, with assignments of the shifted resonances indicated. Combined amide chemical shift perturbations, $\mathrm{\Delta }\delta =\sqrt{\mathrm{\Delta }{\delta }_H^2+{\left(\mathrm{\Delta }{\delta }_N/5\right)}^2}$, are plotted below. The mutation sites relative to FLN5 Y719E have been indicated with star symbols on the CSP plot.

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