Many important biological functions are mediated through protein-ligand interactions and the concomitant conformational change of the protein. The binding of therapeutic agents to the receptor sites of proteins is particularly important in drug design [1-3]. Recent trends in the discovery and development of new medicines demand new methods for rapidly screening protein-ligand binding properties in target selection and lead discovery, as well as for quantifying binding affinities and resolving subtle differences for lead optimization and further development [4]. Although computer modeling has been used to predict binding affinities [2, 5, 6], the strengths of these interactions are normally determined by experimental assays in which either equilibrium titrations, kinetic studies, or stability measurements are involved (e.g. [7-10]). Although success has been achieved in the determination of protein-ligand binding affinities by spectro-scopic, calorimetric and other methods, limitations are often the large amounts of specifically labeled ligand or protein that are required. Often needed are additional spectroscopic or reaction probes, denaturants, or measurements of equilibrium concentrations following a separation, which may perturb the equilibrium. It remains of interest for biochemists and biophysicists to seek additional methods for quantification of protein-ligand binding that have general applicability, high accuracy, relative simplicity, and high throughput.

In the past decade, mass spectrometry has played an important role in the characterization of protein structure, differential expression, dynamics, and functions [11-14]. Several new mass spectrometry-based methods were reported for the characterization of protein-ligand binding [15-21]. Recently, we developed a novel method [22] to quantify protein-ligand interactions in solution by mass spectrometry, titration, and hydrogen/deuterium (H/D) exchange (PLIMSTEX). This strategy can determine the conformational change, binding stoichiometry and affinity in a variety of protein-ligand interactions including those involving small molecules, metal ions, peptides, and proteins [22, 23]. The detailed model ing procedures for the determination of binding constants using PLIMSTEX titration curves and the effect of model modifications on the precision and accuracy were also described [24]. Combined with kinetic measurements of H/D exchange, PLIMSTEX can provide insights on protein structure and protein-ligand interactions, the effect of media and ionic strength [25], species specificity, mutations on protein-ligand binding, and systematic changes in ligands [26]. The determination and interpretation of the titration curves will be described in this chapter. More perspectives of PLIMSTEX and the advantages of this approach over conventional methods and several other mass spectrometry-based methods will be illustrated using several examples.

A complementary approach to PLIMSTEX that we are developing is to compare the reactivity of hydroxyl radicals with a protein-ligand complex to that of the protein alone. If appropriate reagents are used, the change in solvent accessibility or conformation will alter the chemical reactivity of the target, typically the protein, enabling information regarding the location and affinity of the protein-ligand interaction to be determined. Chance and Brenowitz [27] pioneered the use of hydroxyl radicals for modifying Met, Cys or aromatic amino acid residues to determine sites of protein interactions and to follow RNA folding [28] and protein conformational changes [29]. We are developing a faster method that generates and quenches the hydroxyl radicals in less than a microsecond while allowing oxidation of more of the protein's solvent-exposed residues than can be achieved by other methods [30]. An example application shows that apomyoglo-bin has a conformationally flexible F-helix, as also indicated by nuclear magnetic resonance (NMR) [31] and that the porphyrin-binding pocket is closed in the absence of the ligand. Results from using this approach to the S-protein/S-peptide interaction suggest that this method should also be useful for studying binding of protein and ligands.

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