A protein adaptor to locate a functional protein dimer on molecular switchboard

Tien Ngo Anh, Eiji Nakata, Masayuki Saimura, Tsutomu Kodaki, Takashi Morii

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard. We herein report a new adaptor to locate a protein dimer on the DNA origami scaffold based on a homodimeric basic-leucine zipper protein GCN4. Specific binding of GCN4 to programmed addresses on DNA origami and orthogonal targeting by GCN4- and zinc finger protein-based adaptors to the respective addresses on DNA origami were confirmed by gel electrophoretic and AFM analyses. Furthermore, a GCN4-fused homodimeric enzyme showed even higher activity than the wild type enzyme, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami. © 2013 Elsevier Inc.
Original languageEnglish
JournalNature Methods
Volume67
Issue number2
Pages (from-to)142-150
Number of pages9
ISSN1548-7091
DOIs
Publication statusPublished - 2014
Externally publishedYes

Keywords

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology (all)
  • Medicine (all)
  • Atomic force microscopy
  • Basic leucine zipper protein
  • DNA origami
  • DNA-binding protein
  • Self assembly
  • adaptor protein
  • basic leucine zipper transcription factor
  • DNA binding protein
  • nanomaterial
  • single stranded DNA
  • immobilized nucleic acid
  • protein binding
  • amino acid sequence
  • article
  • atomic force microscopy
  • bacterial cell
  • DNA sequence
  • DNA structure
  • Escherichia coli
  • expression vector
  • nanotechnology
  • nonhuman
  • plasmid
  • polymerase chain reaction system
  • priority journal
  • binding site
  • chemistry
  • Basic-Leucine Zipper Transcription Factors
  • Binding Sites
  • Immobilized Nucleic Acids
  • Microscopy, Atomic Force
  • Nanostructures
  • Protein Binding
  • HOMODIMERS
  • enzyme activity
  • DNA binding proteins
  • GCN4
  • zinc finger protein
  • 10060, Biochemistry studies - General
  • AFM analysis laboratory techniques
  • gel electrophoresis electrophoretic techniques, laboratory techniques
  • Biochemistry and Molecular Biophysics
  • Methods and Techniques

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