Mutations at position E174 affect θ222/θ208.

We plotted θ222/θ208, for temperatures from 20 to 92C. Ratios greater than unity typically indicate coiled-coils. θ222/θ208 did not exceed unity for E174A. θ222/θ208 lower than 0.9 indicates helices in isolation [20], and indeed there is no clear coiled-coiling in experimental hGH structures (see PDB ID 1A22). Differences in ratio (comparing WT to E174A and E174Y) are greater at the lower end of the temperature range, presumably due to greater overall order. Linear regressions are shown for all variants (except for L52F, left out for clarity) over the temperature range of 20 to 58C (in which θ222/θ208 varies linearly). Also shown are 95% confidence intervals for the regressions (generated by the seaborn regplot function, in light shading). E174A has clearly higher θ222/θ208 than E174Y and WT. E174Y has somewhat higher θ222/θ208 than WT. L52F is a control, as it is not in a helix and not near the zinc-binding triad; its θ222/θ208 is indistinguishable from that of WT, and is less than that of E174A. E174A / E174Y increase θ222/θ208 ratio, while L52F has no clear effect.