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Figure 4
Characterization of hDHTKD1 disease-causing variants. (a) Weblogo diagrams generated from an alignment of >100 DHTKD1 orthologues and homologues, showing sequence conservation for the regions surrounding the seven missense mutation sites. (b) Small-scale expression and purification for hDHTKD1 WT and variants. SDS–PAGE gel slices showing lysate (L) and eluant (E) samples from affinity purification of hDHTKD145–919 WT, p.S777P and p.L234G. The position of the hDHTKD1 bands is marked by an arrow. Original uncropped gels are shown in Fig. S13 (lanes iv were cropped from one gel and lanes vivii were cropped from another). (c) DSF melting curves for hDHTKD145–919 WT and p.P773L, with an inset showing the derived melting temperature (Tm) values for WT and five variants. (d) Affinity pulldown of hDLST68–453 by immobilized His-tagged hDHTKD145–919 p.R715C variant. The SDS–PAGE shown is one of two biological replicates (Fig. S15). The lanes are loaded with the following samples: 1, flow through; 2–6, wash fractions of increasing imidazole concentration; and 7–11, elution fractions with 250 mM imidazole. (e) hDHTKD1 homodimer showing the sites of the seven missense mutations (black spheres) on one subunit (yellow ribbon). (f)–(h) Atomic environments surrounding the mutation sites (f) p.L234G, (g) p.P773L and p.S777P, and (h) p.R715C. All amino acids shown, including sites of missense mutations, are of the WT protein.

Volume 7| Part 4| July 2020| Pages 693-706
ISSN: 2052-2525