title: Mapping a disordered portion of the Brz2001-binding site on a plant monooxygenase, DWARF4, using a quartz-crystal microbalance biosensor-based T7 phage display.
abstract: In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba 278-8510, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takakusagi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Manita
authors.forename: Tomoe
authors.initials: T
authors.lastname: Kusayanagi
authors.forename: Jesus
authors.initials: J
authors.lastname: Izaguirre-Carbonell
authors.forename: Kaori
authors.initials: K
authors.lastname: Takakusagi
authors.forename: Kouji
authors.initials: K
authors.lastname: Kuramochi
authors.forename: Kazuki
authors.initials: K
authors.lastname: Iwabata
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kanai
authors.forename: Kengo
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Fumio
authors.initials: F
authors.lastname: Sugawara
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000187:drug effects / Q000378:metabolism*; D017123:Bacteriophage T7 / Q000235:genetics*; D001665:Binding Sites; D015374:Biosensing Techniques; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics; D007202:Indicators and Reagents; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012984:Software; D014230:Triazoles / Q000378:metabolism*; D010948:Viral Plaque Assay
chemical_list: C111451:AT3G50660 protein, Arabidopsis; D029681:Arabidopsis Proteins; C451122:Brz2001; D004279:DNA, Viral; D007202:Indicators and Reagents; D019151:Peptide Library; D014230:Triazoles; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases