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WIREs Nanomed Nanobiotechnol
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TOPOFOLD, the designed modular biomolecular folds: polypeptide‐based molecular origami nanostructures following the footsteps of DNA

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Biopolymers, the essential components of life, are able to form many complex nanostructures, and proteins in particular are the material of choice for most cellular processes. Owing to numerous cooperative interactions, rational design of new protein folds remains extremely challenging. An alternative strategy is to design topofolds—nanostructures built from polypeptide arrays of interacting modules that define their topology. Over the course of the last several decades DNA has successfully been repurposed from its native role of information storage to a smart nanomaterial used for nanostructure self‐assembly of almost any shape, which is largely because of its programmable nature. Unfortunately, polypeptides do not possess the straightforward complementarity as do nucleic acids. However, a modular approach can nevertheless be used to assemble polypeptide nanostructures, as was recently demonstrated on a single‐chain polypeptide tetrahedron. This review focuses on the current state‐of‐the‐art in the field of topological polypeptide folds. It starts with a brief overview of the field of structural DNA and RNA nanotechnology, from which it draws parallels and possible directions of development for the emerging field of polypeptide‐based nanotechnology. The principles of topofold strategy and unique properties of such polypeptide nanostructures in comparison to native protein folds are discussed. Reasons for the apparent absence of such folds in nature are also examined. Physicochemical versatility of amino acid residues and cost‐effective production makes polypeptides an attractive platform for designed functional bionanomaterials. WIREs Nanomed Nanobiotechnol 2015, 7:218–237. doi: 10.1002/wnan.1289 This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Protein and Virus-Based Structures
Comparison of the distribution of hydrophobic residues in a natural protein and in a topological protein fold. (a) In the majority of natural protein folds, a single hydrophobic core stabilizes the protein fold. In contrast to native proteins, polypeptide topofolds do not have a discrete hydrophobic core since their structure is defined by the topological arrangement of interacting segments. (b) A large cavity can be observed in the core of such proteins.
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Mathematical topological solutions of self‐assembling tetrahedron from a single chain. Three distinct topomeres built either from four parallel and two antiparallel (a) or three parallel and three antiparallel coiled‐coil pairs (b, c) are possible.
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Topological design of the self‐assembling polypeptide tetrahedron. (a) Twelve coiled‐coil‐forming peptide segments (marked as arrows) were concatenated in a defined order, connected by the tetrapeptide linker segments (SerGlyProGly, blue circle). Four parallel and two antiparallel orthogonal peptide pairs were used to construct a tetrahedron‐forming polypeptide chain based on the publication Gradisar et al. (b) A schematic representation of the polypeptide path forming tetrahedron. (c) Molecular model of the tetrahedral fold. The edges are formed by orthogonal coiled‐coil pairs. (d) Tetrahedral particles visualized by transmission electron microscopy (TEM). Samples of self‐assembled polypeptide were negatively stained after Ni‐NTA‐coated nanogold beads were bound to one vertex of the tetrahedron via the hexahistidine tag. The representative tetrahedron‐like structures from TEM image and projections of a tetrahedron model are presented.
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Coiled‐coil dimer‐based design of topological modular protein folds. (a) Interactions underlying the stability and specificity of a coiled‐coil dimer. The positions of seven amino acid residues (heptad repeat) are denoted by abcdefg. Positions ‘a’ and ‘d’ are typically occupied by hydrophobic residues, forming a hydrophobic core. Positions e and g are frequently occupied by charged residues that participate in the interhelical electrostatic interactions. Positions ‘b’, ‘c’, and ‘f’ can be chemically modified to introduce the desired function into the coiled‐coil assembly. The specificity and orthogonality of the desired coiled‐coil combination can be improved by the negative design, by introducing polar asparagine at the ‘a’ position that most favorably interacts with another Asn at the opposing chain, in order to maximize the difference between the designed and unwanted chain pairing. (b) Self‐assembly of a polypeptide fold based on the concatenated coiled‐coil‐forming segments (green, yellow, violet). Topological fold from a single‐polypeptide chain relies on the specific interactions between concatenated segments to pair in a selected orientation with their complementary interacting segments within the same chain. The topology of the self‐assembled polypeptide chain is defined by the orientation and sequential arrangement of each coiled‐coil pair.
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Characteristic temperature profile of hierarchical DNA nanoassemblies. The most distinguishable feature of hierarchical approaches is the bimodal nature of the first derivative of the temperature profile obtained by thermal annealing. This reflects two thermodynamically uncoupled processes: sequence‐dependent binding of oligonucleotides into structurally defined modules, such as the three‐point star motif (step 2) and their association into more complex assemblies by means of sticky end (step 3) or T‐junction hydrogen bonding.
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Contact map of designed tetrahedron in comparison to folded natural protein. Long‐range interactions occur between the interacting coiled‐coil forming segments of the topological protein fold in this case a tetrahedron (a) in comparison to the natural protein fold(s) comprising primarily β‐strands, in this case cyan fluorescent protein (b), evaluated by the contact map. Parallel and antiparallel orientations of interacting segments can be observed in both maps as lines parallel and perpendicular to the diagonal.
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Strategies for the DNA nanoassembly. (a) Multi‐strand assembly of two‐dimensional (2D) structures using single‐stranded tiles (SSTs). SSTs are color‐coded to present pairwise associations between corresponding binding domains. This does not imply sequence identity. Each SST has a unique sequence and the final structure is fully addressable. Assembly of three‐dimensional (3D) structures was also demonstrated using SSTs. (b) Multi‐strand assembly of a DNA tetrahedron. (c–e) Structural motifs for hierarchical nanoassembly. (c) A paranemic crossover (PX) tile (left) and a double crossover (DX) tile (right). In contrast to PX tiles, DX tiles are topologically entangled. (d) Comparison of an asymmetric Holliday junction (left) and a branched four‐point star motif (right), which offers higher rigidity at the point of branching. (e) A DNA tetrahedron assembled from three‐point star motifs with sticky ends. (f) Scaffold‐based assembly: short oligonucleotides are designed such as to guide the folding of a long single‐stranded DNA into a predetermined planar or 3D object of nanodimensions. (g) Single‐strand assembly approach relies exclusively on intramolecular bonding to guide the folding of a DNA molecule. Models presented in (a) and (c–f) were obtained from the library of parts using NanoEngineer‐1 3D CAD software and the final images were processed using QuteMol molecular visualization system.
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Compact native and modular topological folds of nucleic acids and proteins. Nucleic acids and proteins can form compact folded structures, stabilized by multiple interactions of monomeric units. Both polymers can also be engineered into topological folds defined by the pairwise interactions between modules. Examples of compact native structures and designed modular folds for nucleic acids and proteins are shown. The cobalamin riboswitch aptamer adopts a complex compact tertiary structure (4FRG) (a), stabilized by interactions, similar as the compact fold of a protein, in this example lysozyme (4I8S) (b). A DNA tetrahedron composed by the hierarchical self‐assembly (c) and a single‐chain polypeptide tetrahedron (d) with a hollow core have been designed by the modular approach.
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Number of determined tertiary structures of proteins and number of different protein folds. (a) Growth of the released Protein Data Bank (PDB) structures per year in last two decades http://www.pdb.org/pdb/statistics/contentGrowthChart.do?content=total&seqid=100 (accessed March 28 2014) (dark gray: number of released PDB structures per year, light gray: total number of PDB structures). (b) Data on the growth in the number of folds available in the PDB according to the CATH classification http://www.pdb.org/pdb/statistics/contentGrowthChart.do?content=fold-cath (accessed March 28 2014) (dark gray: number of new folds per year, light gray: total number of unique folds). Growth of the new protein folds stopped in the last years.
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Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures
Biology-Inspired Nanomaterials > Protein and Virus-Based Structures
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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