PLIMSTEX

The first step of PLIMSTEX is a standard continuous labeling HX experiment that can afford kinetic curves, either global or at the peptide level. By comparing the kinetic curves between apo and hard holo (i.e., the protein is completely ligand bound) states, one can determine an HX time point for which the deuterium uptake is nearly constant and there are relatively large differences between the apo and holo states. Under these conditions, HX of the protein and protein-ligand complex are 

D is the deuterium uptake of the apo state protein, and AD are the deuterium uptake differences between the apo state and the intermediates i (i.e., AD. = Dq — D ). Typically, AD is the largest AD, as ligand binding induces protection of the binding sites, so deuterium uptake decreases as hgand is added. D is treated in the modeling as a variable to minimize expaimental errors. The hest fit is obtained by searches, changing all the variable parameters (i.e., D , and AD to minimize the 

The free concentrations are obtained by solving the inverse function of F, shown in Equation 11.10, as one moves along the path 2(r) of the titration experiment  

Solving for F along the path l(j) of total concentrations is facilitated by considering the total derivative with respect to the path variable r as shown in Equation 11.11  

The partial derivatives of F of course, are unknown. But the application of the inverse function theorem [26, 27] allows one to rewrite Equation 11.11 as Equation 11.12  

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M. M. Zhu, R. Chitta, M. L. Gross PLIMSTEX a novel mass spectrometric method for the quantification of protein-ligand interactions in solution. Int. J. Mass Spectrom. 2005, 240, 213—220. 

M. L. Gross Quantification of protein—ligand interactions by mass spectrometry, titration, and H/D exchange PLIMSTEX./. Am. Chem. Soc. 2003, 125, 5252-5253. 

Zhu MM, Hambley D, Gross ML Quantitation of protein-ligand interactions in solution by H/D exchange (PLIMSTEX), chapter 11. 

Quantification of Protein-Ligand Interactions in Solution by Hydrogen/Deuterium Exchange (PLIMSTEX)... 

To validate the method, we applied PLIMSTEX to determine the binding constants (Xa), stoichiometry (1 protein to n ligands), and the protection against H/D exchange in various interactions. We chose as tests the binding of Mg to gua-nosine diphosphate (GDP)-bound human ras protein, of Ca + to apo calmodulin... 

CaM), of fatty acid carboxylate to intestinal fatty acid binding protein (IFABP), and of peptides (e.g, melittin) to Ca +-saturated calmodulin (holo CaM)]. We also extended PLIMSTEX to protein-protein interactions involving self associations of various insulins [33]. These are widely studied systems, and their individual K values range from to 10 M h... 

Fig. n.4 PLIMSTEX curve for 1.5 pM Ras-GDP titrating with Mg +. Conditions 90% D2O, 50 mM HEPES buffer, 100 mM KCI, pH 7.4, H/D exchange time = 3 h. EDTA was used to control [Mg +] in solution. The error bars shown for each data point were based on the deviation from two independent runs. 

Mg +]free was calculated using the Webmaxc Standard program on the internet [43]. The solid line was the fitted PLIMSTEX curve for the average data using a 1 1 binding model and three-parameter (y3i,Do,ADi) fitting. 

Interpretation of PLIMSTEX Results with H/D Exchange Kinetics... 

Application of PLIMSTEX to Relatively Weak Protein-Ligand Binding... 

PLIMSTEX Results for CaM and Intermediate Protein-Ligand Binding Species... 

The PLIMSTEX titration curve shows that CaM becomes more stable (more hydrogen-bonded) upon Ca-binding (Fig. 11.5A). The formation of CaM-4Ca species is the biggest contributor to the shape of the titration curve and accounts for the largest conformational change in the stepwise Ca binding. The earlier... 

PLIMSTEX in Biologically Relevant Media and High Ionic Strength... 

The PLIMSTEX curve for 0.3 pM WT-IFABP titrated with potassium oleate fits well with a 1 1 binding model [22, 24], The K and ADi (difference between the average deuterium level of one-ligand-bound protein and that of apo protein) for WT-IFABP are (2.6 + 0.6) x lO and 13.8 + 0.7 (Table 11.1), respectively, indicating that a strong interaction between oleate and WT-IFABP occurs with protection of 14 backbone amide hydrogens. 

Fig. n.7 Sharp-break PLIMSTEX curves at high protein concentration [23], Line (a) melittin (a 26-amino-acid peptide) titration. Line (b) mastoparan (a 16-amino-acid peptide) titration of 15 pM Ca +-saturated porcine calmodulin (CaM-4Ca) in 50 mM HEPES, 100 mM KCI, 0.49 mM Ca +, 99% D2O, apparent pH 7.4. Data points are based on the average of t /o runs for each titration system, and the breaking point clearly indicates 1 1 protein-ligand binding stochiomet. ... 

PLIMSTEX Curves Linder Different Holo-CaM Concentrations... 

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