TY - BOOK
T1 - Molecular Libraries in Development of Plant Defense Molecules
AU - Madsen, Daniel
PY - 2018
Y1 - 2018
N2 - A double point mutated and destabilized version of the human fkbp12 has previously been engineered to selectively interact with the designed bump-hole ligand shield1. This type of mutated protein is classified as a destabilizing domain (dd) and can confer its instability to any fused protein-of-interest (poi). The utility of the shield1-dd system has enabled researchers to chemically control the presence of any poi in a vast variety of biological systems. With a proposed aim of applying the system on large scale to economically important crops, such as rice and wheat, cheap and scalable alternatives for the synthesis shield1 are in high demand. Using the split-and-mix approach, a one-bead-one-compound library was synthesized on encoded beads with side chain variations on a retained tetrapeptide structure scaffold that had been capped on the n-terminal by a triazole moiety. Computational modeling has been used to design the core structure of the peptide. The library was screened towards a fluorescently labeled version of the dd protein, and the structures of 16 different hit compounds were deconvoluted using an in-house developed decoder apparatus. To verify and distinguish the potency of the solid-supported substrates, the peptides were resynthesized on solid support and screened in a unique on-bead binding assay. Based on the results of the screen, 26 peptides were synthesized on solid support or in solution. The potency of the compounds was tested on arabidopsis thaliana in a plant-based assay, and in vitro by a developed and optimized fluorescence polarization assay. These studies have led to the discovery of a new peptide-based scaffold, and substrates with binding affinities towards the dd in the low micromolar range have been identified.
AB - A double point mutated and destabilized version of the human fkbp12 has previously been engineered to selectively interact with the designed bump-hole ligand shield1. This type of mutated protein is classified as a destabilizing domain (dd) and can confer its instability to any fused protein-of-interest (poi). The utility of the shield1-dd system has enabled researchers to chemically control the presence of any poi in a vast variety of biological systems. With a proposed aim of applying the system on large scale to economically important crops, such as rice and wheat, cheap and scalable alternatives for the synthesis shield1 are in high demand. Using the split-and-mix approach, a one-bead-one-compound library was synthesized on encoded beads with side chain variations on a retained tetrapeptide structure scaffold that had been capped on the n-terminal by a triazole moiety. Computational modeling has been used to design the core structure of the peptide. The library was screened towards a fluorescently labeled version of the dd protein, and the structures of 16 different hit compounds were deconvoluted using an in-house developed decoder apparatus. To verify and distinguish the potency of the solid-supported substrates, the peptides were resynthesized on solid support and screened in a unique on-bead binding assay. Based on the results of the screen, 26 peptides were synthesized on solid support or in solution. The potency of the compounds was tested on arabidopsis thaliana in a plant-based assay, and in vitro by a developed and optimized fluorescence polarization assay. These studies have led to the discovery of a new peptide-based scaffold, and substrates with binding affinities towards the dd in the low micromolar range have been identified.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01011959775&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - Molecular Libraries in Development of Plant Defense Molecules
PB - Department of Chemistry, Faculty of Science, University of Copenhagen
ER -