title: Discovery of light-responsive ligands through screening of a light-responsive genetically encoded library.
abstract: Light-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library. We ligated a photoresponsive azobenzene core to a random CX7C peptide library displayed on the coat protein of M13 phage. A one-pot alkylation/reduction of the cysteines yielded a photoresponsive library of random heptapeptide macrocycles with over 2 × 10(8) members. We characterized the reaction on-phage and optimized the yield of the modifications in phage libraries. Screening of the library against streptavidin yielded three macrocycles that bind to streptavidin in the dark and cease binding upon irradiation with 370 nm light. All ligands restored their binding properties upon thermal relaxation and could be turned ON and OFF for several cycles. We measured dissociation constants, Kd, by electrospray ionization mass spectrometry (ESI-MS) binding assay. For ligand ACGFERERTCG, the Kd of cis and trans isomers differed by 22-fold; an incomplete isomerization (85%), however, resulted in the apparent difference of 4.5-fold between the dark and the irradiated state. We anticipate that the selection strategy described in this report can be used to find light-responsive ligands for many targets that do not have known natural ligands.
authors.affiliation: Department of Chemistry and Alberta Glycomics Centre, University of Alberta , Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Jafari
authors.forename: Lu
authors.initials: L
authors.lastname: Deng
authors.forename: Pavel I
authors.initials: PI
authors.lastname: Kitov
authors.forename: Simon
authors.initials: S
authors.lastname: Ng
authors.forename: Wadim L
authors.initials: WL
authors.lastname: Matochko
authors.forename: Katrina F
authors.initials: KF
authors.lastname: Tjhung
authors.forename: Anthony
authors.initials: A
authors.lastname: Zeberoff
authors.forename: Anastasia
authors.initials: A
authors.lastname: Elias
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
authors.forename: Ratmir
authors.initials: R
authors.lastname: Derda
mesh_terms: D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000737:chemistry* / Q000378:metabolism; D017104:Bacteriophage M13 / Q000737:chemistry*; D008024:Ligands; D008027:Light; D047028:Macrocyclic Compounds / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D055668:Photochemical Processes; D011485:Protein Binding; D019809:Streptavidin / Q000378:metabolism
chemical_list: D001391:Azo Compounds; D008024:Ligands; D047028:Macrocyclic Compounds; D009842:Oligopeptides; D019151:Peptide Library; D019809:Streptavidin; C009850:azobenzene