title: Structure-based design of a photocontrolled DNA binding protein.
abstract: Photocontrolled transcription factors could be powerful tools for probing the role of transcriptional processes in settings that are spatially or temporally complex. We report the structure-based design of a photocontrolled bZIP-type DNA binding protein that is a hybrid of the prototypical homodimeric bZIP protein GCN4 and photoactive yellow protein (PYP), a blue-light-sensitive protein from Halorhodospira halophila. A fusion of the C-terminal zipper region of GCN4-bZIP with the N-terminal cap of PYP was designed based on examination of available crystal structure data, analysis of amino acid preference rules for leucine zippers, and mutational and amino acid conservation data for PYP, together with Rosetta-guided structural modeling. The designed fusion protein GCN4Delta25PYP-v2 is monomeric in the dark; fluorescence, circular dichroism, NMR, and analytical ultracentrifugation data indicate that the zipper domain is hidden. DNA binding in the dark causes substantial structural reorganization of GCN4Delta25PYP-v2 with concomitant slowing of the photocycle, consistent with conformational coupling of the DNA binding domain and the light-sensitive domain of the protein. Consistent with this finding, blue-light irradiation causes a 2-fold increase in specific DNA binding affinity that reverses in the dark. The structure-based approach suggests strategies for enhancing this activity and for producing a family of related photocontrolled proteins for manipulating bZIP activity.
authors.affiliation: Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, Canada M5S 3H6.
authors.forename: Stacy-Anne
authors.initials: SA
authors.lastname: Morgan
authors.forename: Sameer
authors.initials: S
authors.lastname: Al-Abdul-Wahid
authors.forename: G Andrew
authors.initials: GA
authors.lastname: Woolley
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D020607:Halorhodospira halophila / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins