Photo-induced oxidative cross-linking as a method to evaluate the specificity of protein-ligand interactions.
The isolation of protein-binding synthetic molecules from combinatorial libraries or compound collections is now a common practice in chemical biology. An important, but underdeveloped, aspect of characterizing the binding properties of such molecules is their level of binding specificity. This is often evaluated by simply measuring the equilibrium binding affinity of the compound of interest with its target protein and comparing this value with its affinity to one or a few other purified proteins selected at random. These measurements may not reflect accurately the ability of the compound to seek out its target in a complex mixture of proteins such as a cell extract or serum. A more desirable alternative would be to develop solution assays that measure directly the binding of the molecule of interest to both target and competitor proteins in complex solutions. In this report, we evaluate a rapid and efficient photo-triggered cross-linking reaction for assessing binding specificity of synthetic molecules in protein mixtures. Using peptide-protein complexes, we demonstrate that this reaction provides an unbiased view of the peptide-protein contacts present in solution under a given set of conditions and thus is useful for assessing binding specificity. We also discuss the potential application of this chemistry to the related, but more difficult, problem of the identification of protein targets of bioactive molecules.
Lin
H-J
HJ
Center for Biomedical Inventions, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8573, USA.
Kodadek
T
T
D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008024:Ligands; D008027:Light; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*
D003432:Cross-Linking Reagents; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011506:Proteins