Table 4 Validation of different stabilizing effects of ligand binding on thermal stability of dihydrofolate reductases.

From: Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens

Clinically Relevant DHFR Ti (°C)

 

EcDHFR

DfrA1

DfrA5

Ti 1

Δ1

Ti 2

Δ2

Ti

Δ

Ti

Δ

Apo

49.6 ± 0.6

–

60.2 ± 0.3

–

49.8 ± 0.5

–

66.3 ± 0.5

–

+NADPH

53.8 ± 0.4

4.2

63.1 ± 1.3

2.9

56.5 ± 0.8

6.7

69.2 ± 0.5

2.9

+TMP

67.1 ± 2.0

17.5

74.1 ± 0.5

13.9

52.3 ± 0.3

2.5

66.8 ± 0.7

0.5

+NADPH, +TMP

–

–

77.1 ± 0.5

16.9

62.3 ± 0.2

12.5

72.6 ± 0.3

6.3

+UCP1223

68.5 ± 1.4

18.9

80.8 ± 0.2

20.6

58.5 ± 0.1

8.7

67.3 ± 1.0

1.0

+NADPH, +UCP1223

78.9 ± 2.4

29.3

88.3 ± 0.5

28.1

71.2 ± 3.1

21.4

88.5 ± 0.2

22.2

+UCP1228

65.5 ± 4.6

15.9

76.5 ± 2.5

16.3

55.4 ± 1.7

5.6

70. 0 ± 2.7

3.7

+NADPH, +UCP1228

64.3 ± 0.1

14.7

75.7 ± 1.6

15.5

66.5 ± 3.3

16.7

77.3 ± 4.7

11

  1. Thermal stability of EcDHFR, DfrA1 and DfrA5 enzyme as a function of co-factor and inhibitor binding was analyzed by Tycho NT.6 (NanoTemper, Munich, Germany). Temperature-dependent change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm were used to calculate derivatives of the signal (ratio of 350 nm/330 nm). The maximum of the peak corresponds to the inflection point of the underlying ratio curve (inflection temperature, Ti). The gradual stabilizing effect upon ligand binding is reflected in the Ti shift (ΔTi) between the apo form and the corresponding complex. Ti values, with their standard deviations are the averages from at least three independent measurements for each system.
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