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. 2022 May 13;5:459. doi: 10.1038/s42003-022-03384-y

Table 5.

Binding affinity data of NADPH and TMP to EcDHFR, DfrA1 and DfrA5 by microscale thermophoresis (MST).

Complex Titrant KD (nM) Fold change
NADPH KD ± Trimethoprim
EcDHFR apo NADPH 771 ± 145 –
EcDHFR:TMP NADPH 8.1 ± 4.0 95.2 ± 50.3
DfrA1 apo NADPH 1650 ± 230 –
DfrA1:TMP NADPH 430 ± 95 3.8 ± 1.0
DfrA5 apo NADPH 7470 ± 1670 –
DfrA5:TMP NADPH 1540 ± 380 4.9 ± 1.6
Trimethoprim KD ± NADPH
EcDHFR apo TMP 23.6 ± 5.1 -
EcDHFR:NADPH TMP 0.011 ± 0.007 2145.5 ± 1441
DfrA1 apo TMP 27,792 ± 4877 –
DfrA1:NADPH TMP 1308 ± 451 21.2 ± 8.2
DfrA5 apo TMP 11,929 ± 4448 –
DfrA5:NADPH TMP 113 ± 56 105.6 ± 65.5
NADPH KD ± UCP1228
DfrA1 apo NADPH 1044 ± 106 –
DfrA1:UCP1228 NADPH 289 ± 51 3.6 ± 0.7
DfrA5 apo NADPH 6837 ± 1608 –
DfrA5:UCP1228 NADPH 459 ± 157 14.9 ± 6.2

Binding response of the fluorescently labeled dihydrofolate reductases, pre and post incubation step to the increasing concentration of the non-fluorescent ligand. The Data were fitted to a single-site binding model accounting for ligand depletion. Binding constants, KD ± KD confidence (±68% confidence) and the fold change in KD value ± relative error on the fold change were determined from two independent measurements using MO Affinity Analysis v2.1.3 software (NanoTemper Technologies GmbH). Each binding experiment was prepared independently, in parallel using the same preparation of proteins.