title: Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.
abstract: Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.
authors.affiliation: Departments of Chemistry and.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Liu
authors.affiliation: Department of Biochemistry.
authors.forename: Lucas G
authors.initials: LG
authors.lastname: Nivón
authors.affiliation: Department of Biochemistry, Graduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, WA 98195;
authors.forename: Florian
authors.initials: F
authors.lastname: Richter
authors.affiliation: Departments of Chemistry and.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Goldman
authors.affiliation: National Center for Microscopy and Imaging Research, Center for Research on Biological Systems and.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.affiliation: Departments of Chemistry and.
authors.forename: Jennifer Z
authors.initials: JZ
authors.lastname: Yao
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Douglas
authors.initials: D
authors.lastname: Richardson
authors.affiliation: Departments of Chemistry and.
authors.forename: William S
authors.initials: WS
authors.lastname: Phipps
authors.affiliation: Departments of Chemistry and.
authors.forename: Anne Z
authors.initials: AZ
authors.lastname: Ye
authors.affiliation: National Center for Microscopy and Imaging Research, Center for Research on Biological Systems and Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093; and.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.affiliation: Departments of Chemistry and Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Drennan
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, and.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Departments of Chemistry and ating@mit.edu.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D000818:Animals; D055162:Biocatalysis; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D019295:Computational Biology / Q000379:methods*; D003374:Coumarins; D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D008025:Ligases / Q000378:metabolism*; D008854:Microscopy, Electron; D008958:Models, Molecular; D016296:Mutagenesis; D010078:Oxazines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013194:Staining and Labeling
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D010078:Oxazines; D049452:Green Fluorescent Proteins; C014180:resorufin; C030123:coumarin; D008025:Ligases
keywords: LplA; PRIME; chemical probe targeting; enzyme redesign; fluorescence microscopy