Specificity Profiling of Protein‐Binding Domains Using One‐Bead‐One‐Compound Peptide Libraries

Abstract

One‐bead‐one‐compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331‐355 © 2012 by John Wiley & Sons, Inc.

Modulators of Protein–Protein Interactions

“…A neat chemical alternative to the generation and screening of bicylic peptides by phage display is to use the one-bead-two-compound (OBTC) approach, as illustrated by Pei and colleagues . The advantage of OBTC compared to biological display methods is the control over structural diversity: in fact the one-bead-one-compound approach also works well for the generation and screening of nonpeptidic or peptidemimetic compound libraries. − Where OBTC falls down though (and where phage display really excels) is in the evolutionary optimization of active compounds. Nevertheless, the OBTC approach has been applied with success to identify potent inhibitors of Tumor Necrosis Factor-α (TNF-α), for instance Anticachexin C1 ( 53 , Figure ).…”

Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides

“…The screening of an OBOC library often requires a secondary protein that is labeled with a fluorophore or enzyme. For example, secondary antibodies [5] or streptavidin for staining biotinylated proteins have been used [32]. In these cases, false positive peptides might be identified that show affinity to the secondary reagent and not to the target protein.…”

Identification of Nucleophilic Probes for Protease-Mediated Transpeptidation

“…To unbiasedly find a dipeptide nucleophile that efficiently induces transpeptidation, we designed a method using one-bead-one-compound (OBOC) peptide libraries ( Figure 3 ) [ 20 , 21 , 22 ]. The OBOC libraries were composed of beads carrying various peptides in which the N-terminal’s two amino acids (the X 1 and X 2 positions from N-terminus) were randomized and followed by a linker sequence AAARM in which the three alanines, one arginine, and the C-terminal methionine were used for the longer length, solubility and better detection in MALDI, and the cyanogen bromide (CNBr)-induced cleavage of attached peptides, respectively.…”