Identifying Protein-protein Interaction Sites Using Peptide Arrays

Abstract

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein.Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins. Video LinkThe video component of this article can be found at

Peptides derived from the SARS-CoV-2 receptor binding motif bind to ACE2 but do not block ACE2-mediated host cell entry or pro-inflammatory cytokine induction

“…Peptide experiments were performed as described previously [ 21 ]. SARS-CoV-2 receptor binding motif (RBM) peptides were produced by automatic SPOT synthesis [ 22 , 23 ]. Peptides were synthesised on continuous cellulose membrane supports using 9-fluorenylmethyloxycarbonyl chemistry (Fmoc) by the MultiPep RSi Robot (Intavis).…”

High-density arrays to profile circulating biomarkers

“…Peptide microarrays can be used for the same purposes as protein arrays but given the smaller size of peptides, they can yield more information about the specific region of a protein or epitope that interacts with an antibody or with another protein (6). Often this involves the synthesis of overlapping peptides (7) or specific types of protein domains (8).…”

Role of single disulfide linkages in the folding and activity of scyllatoxin-based BH3 domain mimetics

“…Modulators of such protein-protein interactions (PPIs) hold tremendous potential as chemical genetics agents or as therapeutic leads for targeting the enormous number of proteins in the human proteome. Accordingly, there is now significant interest in the development of molecules that can target discrete PPIs with high precision [4][5][6][7]. Owing to large, shallow interaction surfaces, many PPIs have proven difficult or impossible to target using traditional small molecule approaches [8,9], and it is becoming increasingly clear that modulation of such interactions requires molecules that are capable of biomolecular recognition across large surface areas [10,11].…”

“…It has been documented that single disulfides are often difficult to form in peptides and proteins using standard glutathione-based oxidation reactions [36]. However, platinum-based catalysts, such as [Pt(CN) 4 Cl 2 ] 2À and [Pt(en) 2 Cl 2 ] 2+ , have been shown to be powerful and selective oxidizing agents to rapidly form single disulfide bonds in peptides containing up to 20 amino acids [37,38]. We therefore attempted to oxidize our Flu ScTx-Bax ΔΔ peptides using [Pt(en) 2 Cl 2 ] 2+ (Figure 2b).…”