Using the accepted head-to-tail tandem-binding model as a guide, we set out to examine the intermolecular interactions between OmpR dimers bound to DNA by protein protein cross-linking methods.
We determined experimental KD values using a binding model as described in the Methods section of 5.5 ± 1.3 µM for HA, 5.5 ± 1.0 µM for DS, 4.8 ± 0.7 µM for CS4 and 1.4 ± 0.4 µM for CS6 for the GAG hexasaccharides.
The relationship between free and total cefazolin concentrations was best described using a constant binding model as follows: (7) C total = C free f u, where Ctotal represents the total drug concentration and f u represents the (estimated) fraction of free drug.
Hitherto, such atypical kinetic behavior was attributed to a mechanistic scheme involving alternate substrate binding site(s) near the CYP enzyme's active site[11] [14], which is a two-site binding model, as shown in Figure 1.
These data cannot be fit to a single-site binding model as in previous work (9).
Compounds that showed double binding in crystal structures (13, 16, and 17) had their ITCs fitted using a one-site binding model, as this gave the best curve fitting, suggesting the second binder remains relatively weak.
The fraction of unwound duplex was obtained by normalizing the band intensities separately for each gel using the parameters from the fit to a one-site binding model, as the change in fluorescence upon unwinding is different under each condition.
Dissociation constants of JAZ ZF2 and JAZ ZF3 with VAImin RNA were calculated from the changes in the chemical shifts observed in H N HSQC spectra in quantitative NMR titrations using a two-site binding model as previously described.
The results were analysed assuming a one-to-one binding model as previously described [27].
The relationship between FRET efficiency and A/D ratio was modeled with a simple 1∶1 ligand binding model as described before [33], [34].
