The PLIMSTEX curve is directly related to the H/D exchange kinetics of the protein (e.g. ras-GDP) and its protein-ligand complex (e.g. ras-GDP-Mg2+). Taking a certain sampling timepoint (e.g. 3 h) for the titration produces a view that is a weighted superposition of all protein conformation concentrations as a function of ligand concentration. If we were to choose a different time, we would obtain a view with a different weighting. For example, at 3 h, where the H/D exchange becomes nearly constant, the fast and intermediate hydrogens are nearly at equilibrium so their weight in the superposition is approximately zero, but those in the slow group are still exchanging so their weight is dominant. At the start of the titration, ras-GDP is the main species, and it has the smallest number of slow ex changing hydrogens. As a result, we see the highest extent of exchange. At the latter stages of the titration, the ternary complex ras-GDP-Mg2+ is predominant and has the largest population of slow exchanging hydrogens. The middle part of the titration curve (Fig. 11.4) represents a superposition of various ras-GDP and ras-GDP-Mg2+ species. Differences in kinetics give different uptakes of deuterium as a function of sampling time; indeed, if no change in H/D exchange kinetics were observed for different ligand-binding species, the titration curve would be a horizontal line. As mentioned previously, PLIMSTEX is applicable only if there is a change in the D uptake as the ligand is added. When the differences disappear at long time, methods such as pulsed labeling and rapid mixing are better choices because these methods sample fast exchanges
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