Table 1. Overview of the Mutant Variants of DhaA115 Designed and Experimentally Characterized in This Study^a.

software	design	DhaA	mutations	Tmapp [°C]
FireProt	template	115	none	73.3 ± 1.8
DbD	interdomain	181	Y79C, P201C	66.8 ± 0.0
	intradomain	182	I162C, W203C	72.8 ± 1.0
	combined	183	DhaA181 + DhaA182	71.4 ± 1.2
Rosetta	interdomain	184	K74F	62.5 ± 2.5
	interdomain	185	E208Y	66.9 ± 2.0
	interdomain	188	H188W, R204W	70.1 ± 2.7
	intradomain	186	Q150I, I169L	69.6 ± 2.0
	intradomain	187	Q150I, I169L, M186F, N207H	60.1 ± 2.9
	combined	189	Q150I, I169L, M186F, H188W, R204W, N207H	51.2 ± 0.1
FireProt	energy	211	Q165Y, G171I, A212N	75.3 ± 1.3
	ratio	212	R30G, V184E, V197E, N217D	76.9 ± 0.6
	ratio*	213	DhaA212-R30G	77.0 ± 0.7
	majority	214	N119H, L161M, I163L, W203L, E285A	64.0 ± 0.9
	majority*	215	DhaA214-N119H, W203L, E285A	67.3 ± 1.0
	combined	216	DhaA211 + DhaA213 + DhaA215	72.8 ± 0.9
	combined*	223	DhaA211 + DhaA213	79.3 ± 0.9
PROSS	design 2	217	D11P, R30P, N119H, K124R, V184E, V197E, N217D, N227R, H230A, T242E, S258W	77.0 ± 0.7
	design 2*	218	DhaA217-R30P, N119H, H230A, S258W	77.3 ± 1.5
	design 5	219	DhaA217 + D156G, F205W, A212D, I218V, L221I, R254W, K263E, S284R	73.8 ± 0.7
	design 5*	220	DhaA219-F205W, R254W, K263E, S284R	76.6 ± 1.8
	design 8	221	DhaA219 + G7S, S58T, D82E, E99D, K117R, Q150K, E160K, L161M, D164E, A167M, G171Q, K175A, A250E, A253E, L255C, A256R, L281E	78.4 ± 0.9
	design 8*	222	DhaA221-R30P, N119H, H230A, S258W, F205W, R254W, K263E, S284R	80.9 ± 0.7
PROSS, MutCompute		231	DhaA222-D11P, A212D, and L221I	81.7 ± 0.9

^aThe asterisk denotes variants in which mutations designed by the automated tools have been removed based on manual curation described in the text (bold). The values of T_m^app are means and standard deviations obtained from three different techniques: DSC, CD, and differential scanning fluorimetry (DSF), except for variants containing disulfide bridges where the values represent means and standard deviations of T_m^app from three replicates of DSF measurements.