Abstract
Nano-scale alignment of several proteins with freedom of motion is equivalent to an enormous increase in effective local concentration of proteins and will enable otherwise impossible weak and/or cooperative associations between them or with their ligands. For this purpose, a DNA backbone made of six oligodeoxynucleotide (ODN) chains is designed in which five double-stranded segments are connected by four single-stranded flexible linkers. A desired protein with an introduced cysteine is connected covalently to the 5′-end of azido-ODN by catalyst-free click chemistry. Then, six protein-ODN conjugates are assembled with their complementary nucleotide sequences into a single multi-protein-DNA complex, and six proteins are aligned along the DNA backbone. Flexible alignment of proteins is directly observed by high-speed AFM imaging, and association of proteins with weak interaction is demonstrated by fluorescence resonance energy transfer between aligned proteins.
Highlights
Protein-protein interactions play a critical role in numerous biological processes, and understanding the nature of each interaction is of central importance in biology and biotechnology
We designed a DNA backbone composed of five solid segments, each connected by a flexible linker (Fig. 1A)
The hybridized structure contains five double-stranded DNA segments connected by four single-stranded trithymidylate regions
Summary
Protein-protein interactions play a critical role in numerous biological processes, and understanding the nature of each interaction is of central importance in biology and biotechnology. Interactions are transient or weak, producing a complex that is difficult to isolate To study such complex, component proteins of the complex are chemically crosslinked or genetically fused as a single polypeptide to prevent the complex from dissociation. Component proteins of the complex are chemically crosslinked or genetically fused as a single polypeptide to prevent the complex from dissociation We report a procedure to align several different proteins, all connected to a single flexible DNA backbone. This increases local concentrations of the proteins and will enable association of proteins with very weak interactions
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