Table 3. Gibbs free unfolding energy of HET-s(218–289) amyloid fibrils.

Variant	ΔG(a) (kcal/mol)	ΔΔG (kcal/mol)	m-value	Reproducibility and denaturation curve shape when compared to wt	In vivo [Het-s] Property ([Het-s] activity/propagation rate/spore killing (Figure 2; Tables 1–2)
WT	2.9±0.2		wt	y, wt
K229A	4.4±0.0	1.5±0.2	wt	y, wt-like	wt-like
I231A	n. d.	n. d.	not wt	y, no plateau at low GuHCl conc.	[Het-s] reduced
V239A	3.9±0.5	1±0.5	wt	y	wt-like
Q240A	n. d.	n. d.	not wt	n, no plateau at low GuHCl conc., not a two-state process	[Het-s] reduced/reduced propagation and spore killing
L241A	3.9±0.1	1.0±0.3	wt	y, wt-like	wt-like
N262A	n. d.	n. d.	not wt	y, not a two-state process	[Het-s] reduced
V264A	4.0±0.2	1.0±0.3	wt	y, wt-like	wt-like
V267A	2.9±0.1	0.2±0.3	wt	y, wt-like	wt-like
E272A	4.8±0.1	1.9±0.3	wt	y, wt-like	wt-like/reduced propagation
S273A	4.3±0.1	1.4±0.2	wt	y, wt-like	wt-like
G278A	2.5±0.3(b) 1.3±0.1(b)	-0.8±0.4	not-wt	n, no plateau at low GuHCl conc.	[Het-s] strongly reduced
F286A	n.d	n.d	not wt	y, not a two-state process	[Het-s] strongly reduced
D288A	4.1±0.1	1.2±0.3	not wt	y, wt-like	[Het-s] reduced/enhanced prion formation
F286A/W287A	n.d	n.d	not wt	y, not a two-state process	n.d.

^(a)The ΔG value was obtained using the m-value of the wt unless stated in the fourth column that the curve fitting was improved with a free m-value different from the corresponding wt one.

^(b)The ΔG value was obtained using a free m-value. Two different denaturation curves were obtained and their individual ΔG values are listed.