To validate the method, we applied PLIMSTEX to determine the binding constants (Ka), stoichiometry (1 protein to n ligands), and the protection against H/D exchange in various interactions. We chose as tests the binding of Mg2+ to gua-nosine diphosphate (GDP)-bound human ras protein, of Ca2+ to apo calmodulin
(CaM), of fatty acid carboxylate to intestinal fatty acid binding protein (IFABP), and of peptides (e.g., melittin) to Ca2+-saturated calmodulin (holo CaM)]. We also extended PLIMSTEX to protein-protein interactions involving self-associations of various insulins . These are widely studied systems, and their individual K values range from 104 M_1 to 108 M_1.
Modeling the titration results for the test systems (Table 11.1) gives the binding stoichiometry from the best fit. The binding constants determined by PLIMSTEX for the tested system are within a factor of six of those reported previously using established methods (Table 11.1). The positive AD; values (Table 11.1) quantify the increased hydrogen bonding (more protection to H/D exchange). The AD; values in the case of insulin represent changes in the solvent accessibility of the
Table 11.1 Outcome of test systems for PLIMSTEX.
Protein (Ctota|) + ligand AD¡lal PLIMSTEX Ka (MC1 )[a] Ka(Literature)
r-Human insulin + r-human insulin (mono- to di- to hexamer)
K3: (7 G 2) x 104; K4: (1.1 G 0.4) x 105; K3K4: (9 G 1) x 109
a Each protein-ligand titration was done in duplicate. Values were determined by fitting the average data at similar conditions. A subsampling method was used to evaluate the second order statistics of the parameters.
b Kj (literature data) determined under comparable experimental conditions (e.g., similar pH, ionic strength if available) were selected. c DDi.
fFrom reference . g From reference .
hFrom reference  for CaM from bovine brain.
1 From reference  for CaM from wheat germ. ' AD12. k AD26.
1 From reference .
oligomer compared to that of monomer. Each system is now discussed in more detail.
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