Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.
Structural DNA nanotechnology [1,2] and the technique of DNA origami [3] enable the rapid generation of a plethora of complex self-assembled nanostructures. [4][5][6] Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years.[7] The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibodyantigen interactions, [8,9] aptamer binding, [10,11] nucleic acid hybridization of DNA-tagged proteins, [12,13] or predominantly biotin-streptavidin (STV) interactions. [14][15][16][17][18][19] We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin-STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O 6 -alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as "Snap-tag", [20] or haloalkane dehalogenase, which is also known as "HaloTag".[21] By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins.To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, [20] and the commercially available HaloTag [21] system. The respective smallmolecule suicide tags (O 6 -benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1 a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1 b). To gain access to fusion proteins bearing the complementary Snap-and Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) [22] and mKate, [23] the enzymes cytochrome C peroxidase (CCP) [24] and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), [25] to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein...
Structural DNA nanotechnology [1,2] and the technique of DNA origami [3] enable the rapid generation of a plethora of complex self-assembled nanostructures. [4][5][6] Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years.[7] The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibodyantigen interactions, [8,9] aptamer binding, [10,11] nucleic acid hybridization of DNA-tagged proteins, [12,13] or predominantly biotin-streptavidin (STV) interactions. [14][15][16][17][18][19] We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin-STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O 6 -alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as "Snap-tag", [20] or haloalkane dehalogenase, which is also known as "HaloTag".[21] By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins.To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, [20] and the commercially available HaloTag [21] system. The respective smallmolecule suicide tags (O 6 -benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1 a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1 b). To gain access to fusion proteins bearing the complementary Snap-and Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) [22] and mKate, [23] the enzymes cytochrome C peroxidase (CCP) [24] and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), [25] to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein...
The development of nanobiotechnological devices requires the ability to build various components with nanometer accuracy. DNA is a well-established nanoscale building block that self assembles due to specific interactions that are encoded in its sequence. Recently, it has become possible to couple proteins to DNA, thereby expanding the capabilities of DNA for use with molecular photonics and bioelectronics. Here, we present the design and characterization of a supramolecular Förster resonance energy transfer (FRET) system by using a fluorescent protein bound to single-stranded DNA (ssDNA), a fluorophore attached to a second ssDNA molecule, and a complementary strand for hybridizing the two fluorophores together. The FRET efficiency was studied by using both ensemble and single-pair FRET measurements. The distance between the two fluorophores was determined from the single-pair FRET efficiency and could be described by a simple cylindrical model for the DNA. Hence, DNA can be used as a scaffold for positioning fluorescent proteins, as well as traditional fluorophores, with nanometer accuracy and shows great potential for use in the future of nanobiotechnology.
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