Table 1.

Binding constants and probability estimation of PDZ-mutants and peptide ligand

PDZKD,* μMkon, μM−1·s−1ΔΔΔGC, kcal·mol−1Probability § ΔΔGD-N, kcal·mol−1
WT0.5 (0.1)10 (0.3)
F325A0.310 (0.2)3.0 (0.1)
V328A1.7 (0.1)9 (0.1)0.2 (0.2)
G329A46 (4)9 (0.7)0.5 (0.1)
F340A1.010 (0.1)0.2 (0.1)
I341V0.510 (0.1)−0.2 (0.1)
V362A0.7 (0.1)7 (0.1)0.7 (0.1)
A376G2.5 (0.7)7 (0.1)1.0 (0.1)
A376V0.3 (0.02)8 (0.1)−0.7 (0.1)
K380A1.5 (0.1)7 (0.1)−1.0 (0.1)
V386A0.58 (0.1)1.2 (0.1)
A390G0.410 (0.1)2.2 (0.1)
H372Y67 (4)nd −2.1 (0.1)
F325A/H372Y28 (8)nd0.30.793.6 (0.1)
V328A/H372Y330 (130)nd−0.2 (0.5)0.380.4 (0.1)
G329A/H372Y490nd1.5−0.1 (0.1)
F340A/H372Y98nd0.20.780.0 (0.1)
I341V/H372Y70nd0.00.600.1 (0.1)
V362A/H372Y36nd0.60.951.8 (0.1)
A376G/H372Y100 (5)nd0.7 (0.4)0.971.5 (0.1)
A376V/H372Y48nd−0.2−1.0 (0.1)
K380A/H372Y150 (6)nd0.2 (0.2)0.76−0.8 (0.1)
V386A/H372Y79 (21)nd−0.10.541.7 (0.1)
A390G/H372Y54nd−0.10.532.6 (0.1)
  • *K D values were obtained by fitting Eq. 1 to the equilibrium binding data. Errors are shown in parenthesis and were calculated from at least three independent measurements.

  • The association rate constant k on was determined by fitting Eq. 2 to the plots of observed rate constants versus PDZ concentrations. Errors shown in parenthesis are fitting errors.

  • ΔΔΔG C is the coupling energy for ligand binding between tested positions and H372. Errors shown in parenthesis are propagated errors in the K D values.

  • §The estimate of probability ΔΔΔG C > 0 is based on the posterior distribution of the mean among one to five measurements of independent K D measurements. The model fitted is described in Materials and Methods.

  • ΔΔΔG D-N is the difference in stability between the wild-type and the single mutants or between the H372Y mutant and the double mutants. Errors shown in parenthesis are estimated from fitting errors of the denaturation constants.

  • nd (not determined), no association rate constants were determined for the double mutants. Observed rate constants were too high for the stopped-flow machine because of large off-rate constants.