title: Novel peptide-protein assay for identification of antimicrobial peptides by fluorescence quenching.
abstract: The specific interaction of peptides with proteins is often a key factor which determines biological activities. The determination of K(d) values of such interactions is commonly performed with fluorescence polarization. However, fluorescence polarization assays are prone to false-positive results due to the potential for non-specific interactions and only afford very low signal-to-background ratios. Here, we present as an alternative a fluorescence resonance energy transfer based quenching assay to measure peptide-protein interactions in solution. In a test setup where antimicrobial peptides were tested for their affinity towards the protein DnaK, the assay provided high specificity and good reproducibility and correlated with the results obtained by fluorescence polarization methods. Furthermore, we established a fast prescreening method which will allow a highly efficient screening of peptide libraries by reducing the amount of sample by 98% compared to conventional fluorescence polarization assays.
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Center of Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Dobslaff
authors.forename: T
authors.initials: T
authors.lastname: Kreisig
authors.forename: N
authors.initials: N
authors.lastname: Berthold
authors.forename: R
authors.initials: R
authors.lastname: Hoffmann
authors.forename: T
authors.initials: T
authors.lastname: Zuchner
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000378:metabolism*; D001419:Bacteria / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000191:economics / Q000379:methods*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins