Exploring Peptide Space for Enzyme Modulators

Abstract

Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry.Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis.Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening highdensity arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and β-galactosidase (β-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays.Several peptides, selected from microarrays, were able to inhibit β-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation.PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover ii molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry.

Unstructured interactions between peptides and proteins: Exploring the role of sequence motifs in affinity and specificity

“…Even the transfer of substrates between enzymes can utilize unstructured interactions to keep the local concentration of product/substrate high and facilitate transfer [11]. Indeed, protein interactions in biology almost certainly represent a continuum between structurally defined interactions with long residence times and transient unstructured interactions.…”

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

“…Combinatorial peptide selection can generate not only material-binding peptides, but also peptides having affinity for certain proteins, such as Fc region of IgG [211–213] and enzymes (namely horseradish peroxidase, alkaline phosphatase and β-Gal β-galactosidase) [214–216]. Jung et al demonstrated that highly oriented layer of antibodies was obtained when immobilized on gold SPR chips coated with Fc-specific peptides.…”

Microarray Selection of Cooperative Peptides for Modulating Enzyme Activities

“…Microarrays of 10,000 20-mer peptides were produced by Arizona State University as previously reported [14,17]. Briefly, 10,000 distinct polypeptide sequences were spotted in duplicate onto a glass slide possessing an amino-silane surface coating.…”

“…These arrays can be made by printing pre-synthesized peptides [14], SPOT synthesis (spotting and synthesis) [15], and light-directed, spatially-addressable synthesis [16]. Hydrogel-coated peptide microarrays were also reported as a means of screening for enzyme activity that is modulated by specific protein–peptide interactions, and has made it possible to perform activity assays using high-density microarrays [17]. …”