title: Specific labeling of zinc finger proteins using noncanonical amino acids and copper-free click chemistry.
abstract: Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays, and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Younghoon
authors.initials: Y
authors.lastname: Kim
authors.forename: Sung Hoon
authors.initials: SH
authors.lastname: Kim
authors.forename: Dean
authors.initials: D
authors.lastname: Ferracane
authors.forename: John A
authors.initials: JA
authors.lastname: Katzenellenbogen
authors.forename: Charles M
authors.initials: CM
authors.lastname: Schroeder
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016335:Zinc Fingers
chemical_list: D000596:Amino Acids; D017931:DNA Primers; D003300:Copper