Table 1. Protonation states of Glu68 and Asp234.

Changes in the protonation states are in bold. – indicates not applicable.

		Fixed state during MD*	Calculated state after MD†
Wild-type (1)	E68	Protonated	Protonated
	D234	Deprotonated	Deprotonated
Wild-type (2)	E68	Protonated	Protonated
	D234	Protonated	Deprotonated
Wild-type (3)	E68	Deprotonated	Protonated
	D234	Protonated	Deprotonated
E68D (1)	D68	Protonated	Protonated
	D234	Deprotonated	Deprotonated
E68D (2)	D68	Deprotonated	Protonated
	D234	Deprotonated	Deprotonated
E68Q (1)	Q68	–	–
	D234	Deprotonated	Deprotonated
E68Q (2)	Q68	–	–
	D234	Protonated	Deprotonated
D234E (1)	E68	Protonated	Protonated
	E234	Deprotonated	Deprotonated
D234E (2)	E68	Protonated	Protonated ‡
	E234	Protonated	Protonated ‡
D234N (1)	E68	Deprotonated	Deprotonated
	N234	–	–
D234N (2)	E68	Protonated	Protonated §
	N234	–	–
E68Q/D234N	Q68	–	–
	N234	–	–

^*The system was equilibrated for 5 ns. 10 conformations were sampled at 0.1 ns intervals during the 1 ns production run.

^†Protonation patterns obtained using the MD-generated conformations.

^‡Although we were able to obtain the MD-generated conformation with protonated Glu68 and protonated Glu234, which was confirmed in the calculated protonation pattern, the conformation cannot reproduce the experimentally measured absorption wavelength (Supplementary file 1D) and is unlikely relevant to the D234E GtACR1.

^§Although we were able to obtain the MD-generated conformation with protonated Glu68, which was confirmed in the calculated protonation pattern, the protonation state is not consistent with deprotonated Glu68 suggested in FTIR studies by Dreier et al., 2021.