Chemical Biology : Peptides and peptide arrays

Peptide Chemistry STP

Peptide arrays

The STP has expertise in the design and synthesis of a range of peptide-based tools and membrane supported peptide arrays. This expertise can be applied to address a range of biological questions, from the identification of highly selective peptidic modulators of a protein target, through to using peptide arrays to probe the protein sequences important in determining the affinity of protein-protein interactions.

Peptide tools

The group designs and synthesises linear, branched, cyclic and disulphide bridged peptides, especially those with complicated modifications, including fluorophores, chemical handles to enable cross-linking and post-translational modifications such as lipidation and phosphorylation.

Peptide arrays

Many protein-protein interactions can be studied using a synthetic peptide as one of the partners. To enable the exploration of such interactions in a high-throughput manner, we make peptide arrays on cellulose membranes, which can be probed by an interacting protein to allow the study of molecular recognition events.

As well as defining binding sites between protein partners, peptide arrays can also be useful in studying enzyme-substrate interactions or antibody epitopes.

Peptides are synthesised on a cellulose sheet in spots of around 3 mm diameter, each containing about 20 nmol of peptide. These peptides have their C-terminus anchored to the cellulose, with the acetylated N-terminal projecting out from the sheet.

A protein can be scanned across its length by making 16-25 mers shifted by one amino acid, then looking for the site of interaction with its protein partner. For example, in Molecular Cell, 2022, 82, 4324–4339 a series of peptide arrays of protein ATG2A were synthesised to explore the structural drivers of ATG2A’s interaction with its binding partner ATG9A (see images below).

close-up of a protein structure next to peptide arrays

Peptide array of “Fragment 4” of ATG2A incubated with ATG9A. The array identified two putative ATG9A binding sites that have a Gaussian-like distribution of binding.

Once a binding site has been found using a peptide array, it is often validated by synthesising the interacting peptide(s) to perform pulldown or inhibition-of-binding assays. 

Once binding sites are validated, mutation arrays may be synthesised in which every amino acid in a sequence is substituted by every other amino acid. This allows detection of the amino acids that are most important for a particular protein-protein interaction.