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Protease interaction studies

Page Contents
Applications Using FRET PepSets
REPLi: a tool for rapidly identifying protease substrates
FRET Peptide Library Design
Common FRET pair examples
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Fluorescent Resonance Energy Transfer (FRET) is commonly used to investigate protein interactions such as those involving proteases. Using a unique parallel synthesis technology, PepSets peptide libraries can be prepared to individual specifications quickly and affordably using a range of labels suitable for use in FRET peptides


Applications Using FRET PepSets

PepSets peptides are frequently tagged so that their interactions with other molecules can be monitored. When labelled with two dyes, which have overlapping spectra, peptides can be used in fluorescence resonance energy transfer (FRET) assays.

A FRET peptide
FRET peptides contain a fluorescent donor and acceptor group. When the peptide is cleaved, the fluorophore begins to fluoresce, indicating that enzyme activity was not inhibited.

FRET peptides are typically labelled with a fluorescent donor and a non-fluorescent acceptor. When these molecules form part of the same short peptide, they are close enough for the acceptor to quench the signal from the donor and for there to be very little fluorescence emission. However, if the acceptor is removed - for example, by protease cleavage - the quenching effect is lost, leading to increased fluorescence from the donor at the appropriate excitation wavelength.

FRET peptides can therefore be used to study any biochemical reaction, which changes the physical distance between donor and acceptor molecules. FRET assays offer a safer alternative to the use of radiolabelled isotopes and methods are quick, sensitive and easily automated.


Protease assay using FRET peptides

REPLi: a tool for rapidly identifying protease substrates

Identification of peptide substrates for proteases can be a major undertaking. To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, Mimotopes has developed in collaboration with GSK a ‘small but smart’ generic fluorescence resonance energy transfer rapid endopeptidase profiling library (REPLi) as a tool for rapidly identifying protease substrates. Within a tripeptide core, flanked by Gly residues, similar amino acids were paired giving rise to a relatively small library of 3375 peptides divided into 512 distinct pools each containing only 8 peptides. The REPLi has been validated with trypsin, pepsin, the matrix metalloprotease (MMP)-12 and MMP-13 and calpains-1 and -2. In the case of calpain-2, a single iteration step involving LC-MS, provided the definitive residue specificity from which a highly sensitive fluorogenic substrate was then designed. The thorough validation of this ‘small but smart’ peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases.

PepSets REPLi is presynthesized in two scales (5nmol and 50nmol) and ready to ship. View REPLi specifications in further detail.


Tripsin Assay with FRET peptides
A combinatorial library of FRET peptides was used to obtain kinetic data on protease specificity. This figure illustrates the increase in fluorescence with time, resulting from peptide cleavage, for 96 peptides from the library.

FRET Peptide Library Design

Fluorescent donor and acceptor groups are quite large so spacer residues are usually incorporated into test sequences to ensure access to potential recognition sites. Hydrophilic spacers, such as b-alanine, are preferred as these make the peptides easier to solubilise. If a hydrophobic spacer has to be used, peptide solubility can be improved by the addition of one or more lysine residues at the C terminus.

Synthesis procedures are monitored closely to minimise the incidence of peptides labelled with only one or other of the fluorophores.

FRET peptides can be designed with or without prior knowledge of target sequences. Where information about targets is limited, a replacement set of amino acids can be defined for each residue position and all possible combinations prepared. If the replacement set is large or two or more residue positions are variable, mixtures can be used to reduce the number of peptides in the screening set.

Replacement sets can include L-amino acids, D-amino acids and any unusual amino acids, which are available commercially in an Fmoc-protected form.


Common FRET pair examples

Examples of common FRET pair examples are shown here, but many others are possible. Investigate some of the fluorescent labels Mimotopes provides.

Common FRET pair examples

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