0 × 10-7 M. These values indicate that the two best YbaBHI binding sites on this DNA are of nearly equal affinity; the ~2-fold difference in affinity between first and second binding steps is just what would be expected on a statistical basis for independent binding to identical sites [13]. Parallel measurements were made for the binding of YbaBEc to the b-WT DNA fragment

(Fig. 4B). These data also indicate that 2 molecules of YbaBEc bound free DNA to form the first complex and two more bound to form the second complex. The association constants for the first and second binding steps are Ka,1 = 1.7 ± 0.8 × 1014 M-2 and Ka,2 = 2.9 ± 0.5 × 1013 M-2. Assuming equipartition of binding free energies as before, these correspond to monomer-equivalent dissociation constants Kd,1 = 7.7 ± 0.4 × 10-8 M and Kd,2 = 1.9 ± 0.3 × 10-7 M. As with the H. influenzae protein, the ~2-fold difference in affinity is what would MLN0128 molecular weight be expected for independent binding to two identical sites. We note that these binding

constants reflect binding under our standard in vitro conditions and should not be interpreted to represent the corresponding affinities DAPT datasheet for binding in vivo. None of our binding data suggests that either protein can bind DNA as a monomer. YbaBHi and YbaBEc proteins crystallized as dimers, and both previous sedimentation analyses and our gel filtration analyses indicated that YbaBHi exists primarily as a homodimer in solution [data not shown and [3]]. Taken together, these data indicate that the homodimer is the basic unit of DNA-binding activity for this family of proteins. Figure 4 Analysis of

stoichiometries and affinities of YbaB Ec and YbaB Hi binding to b-WT DNA. Data from the experiments shown in Fig. 3. (A) Binding of YbaBEc. Symbols: (black circle), first binding step; (black square), second binding step. The lines are least-squares fits to Eqs 4 and 5, returning stoichiometry values of 1.93 ± 0.14 Lepirudin for the first binding step and 2.16 ± 0.14 for the second. From the logarithm of the free protein concentration at the midpoint of each binding transition we estimate that Ka,1 = 1.7 ± 0.8 × 1014 M-2 and Ka,2 = 2.9 ± 0.5 × 1013 M-2. The ranges given for these parameters are 95% confidence limits calculated for the least squares fits. (B) Binding of YbaBHi. Symbols: (black circle), first binding step; (black square), second binding step. The lines are least-squares fits to Eqs 4 and 5, returning stoichiometry values of 2.09 ± 0.16 for the first binding step and 2.18 ± 0.19 for the second. From the logarithm of the free protein concentration at the midpoint of each binding transition we estimate Ka,1 = 1.7 ± 0.7 × 1013 M-2 and Ka,2 = 3.0 ± 1.4 × 1012 M-2. The ranges given for these parameters are 95% confidence limits calculated for the least squares fits. In control experiments, purified YbaB proteins were treated either by incubation with 1 mg/ml proteinase K for 30 min or by heating in a boiling water bath for 10 min.