Table 3.

Experimental and predicted solubility of ThreeFoil variants

Values for each solubility prediction tool were normalized to be 1.0 for WT. In cases where the tool's output is a lower value when solubility is decreased, the score is simply normalized as Mut_score /WT_score. In cases where the tool's output is a higher value when solubility is decreased, the normalized score is (1/Mut_score)/(1/WT_score) (alternatively, WT_score/Mut_score). Thus, all predictions of reduced solubility relative to WT have scores < 1.0. The following output values were used from each tool: Aggrescan3D (99), total score; CamSol (100), intrinsic solubility; PASTA (101), number of amyloid-forming regions; TANGO (102), Agg parameter; ZipperDB (103), number of amyloid-prone regions; Zyggregator (104), intrinsic aggregation propensity. Hydrophobicity is defined by the change in side chain solvation free energy, as determined by Wimley et al. (65).

Mutant	Solubility	Aggrescan3D	CamSol	PASTA	TANGO	ZipperDB	Zyggregator	Hydrophobicity
	mg/ml
WT	23.8	1.00	1.00	1.00	1.00	1.00	1.00	1.00
K53V	22.5	0.97	0.78	0.98	0.01	0.94	1.08	0.95
A62V	21.1	1.00	0.98	1.00	1.00	0.88	0.99	0.98
D85P	16.9	0.96	0.99	1.00	0.99	1.00	0.92	0.94
D49N	25.0	0.98	0.99	1.00	1.00	0.94	0.91	0.95
A68G	21.9	1.00	1.01	1.00	1.00	1.00	1.00	1.01
R90L	16.8	0.92	0.87	1.00	0.42	1.00	1.08	0.95
D93P	21.2	0.93	0.98	1.00	1.00	1.00	0.92	0.94
E66Y	19.9	0.95	0.98	1.00	1.00	1.00	0.90	0.93
E66L	17.0	0.96	0.99	1.00	1.00	1.00	0.90	0.92
Q78I	1.4	1.00	0.96	0.91	0.02	1.00	0.99	0.97
MMut1	0.2	0.79	0.28	0.86	0.00	0.63	1.01	0.72
MMut2	0.1	0.76	0.15	0.86	0.01	0.58	0.73	0.65
R		0.71	0.75	0.93	0.75	0.69	0.30	0.75
ρ		0.60	0.63	0.58	0.64	0.20	0.15	0.59