Fig. 3: Rational design of PAS domain (PASD) suppressor variants.

(a) Structure of the Kv11.1 PASD (PDB: 1BYW) showing the location and (b) conservation of each variant characterized. (c) FoldX calculated stabilities for increasingly larger hydrophobic substitutions and V41 and C64 variants. Bars ± SD represent ΔΔG predictions from three different structures (PDB: 1BYW, 4HQA, 4HP9).²³ (d) Representative western blot of each full-length Kv11.1 variant expressed in HEK cells under normal conditions (-) or at reduced temperature (27 °C). A lack of a 155-kD bands indicates defective trafficking. (e) Representative immunoblots of recombinant PASD C64 and V41 hydrophobic substitutions expressed in E. coli. Bars ± SD represent relative solubility (% of wild type [WT]) determined by dot blot (n ≥ 3). (f) Changes in FoldX stabilities are shown for second-site variants predicted to improve variant stability (smaller ΔΔG). Representative western blots of each full-length Kv11.1 variant expressed in HEK cells are also shown under normal conditions (-) or at reduced temperature (27 °C). (g) Representative immunoblots of recombinant PASD suppressor variants expressed in E. coli. Bars ± SD represent relative solubility (% of WT) determined by dot blot (n ≥ 3). (h) Current densities and western blots for full-length Kv11.1 variants expresses in HEK cells. Inset shows voltage-clamp protocol with representative current trace. Bars ± SD represent current density levels (n ≥ 4 cells). Dashed line indicates WT current density level previously reported,¹⁶ which was performed at the same time and with the same intracellular and extracellular solutions as these variants. *P < 0.05, a significant reduction in solubility of variants compared with WT (e), increase in solubility of the double variant compared with the single variant (g) or increase in current density of the double variant compared with the single variant (h).