title: Complete substitutional analysis of a sunflower trypsin inhibitor with different serine proteases.
abstract: Here we present a method to simultaneously characterize and/or optimize both the binding loop towards the protease and a cysteine-stabilized scaffold. The small peptidic sunflower trypsin inhibitor (SFTI-1) was chosen as a model system for these experiments. The inhibitor was investigated for positional specificity against trypsin, elastase and proteinase K using complete substitutional analyses based on cellulose-bound peptide spot synthesis. Inhibitor variants optimized for elastase or proteinase K inhibition by several rounds of substitutional analyses exhibit K(i) values in the micromolar range and high specificity for the corresponding protease. The results of this easy-to-perform assay can be used to design an improved peptide library using classical methods.
authors.affiliation: Department of Biochemistry, Medical Faculty Charité, Humboldt University of Berlin, Monbijoustrasse 2, 10117 Berlin, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Hilpert
authors.forename: Gerd
authors.initials: G
authors.lastname: Hansen
authors.forename: Helga
authors.initials: H
authors.lastname: Wessner
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer-Engert
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Höhne
mesh_terms: D000595:Amino Acid Sequence; D019286:Endopeptidase K / Q000378:metabolism; D006368:Helianthus / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000187:drug effects*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D019151:Peptide Library; D010456:Peptides, Cyclic; D010940:Plant Proteins; D011480:Protease Inhibitors; C448501:SFTI-1 peptide, sunflower; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; D014357:Trypsin; D019286:Endopeptidase K