Chemically Controlled Self-Assembly of Protein Nanorings

Nucleic Acids Research, 1994

Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays.

Methods in molecular biology (Clifton, N.J.), 2011

Continued exploration into the field of chemically induced dimerization (CID) has revealed a number of applications for its use in a broader context as a method of structural assembly (1-4). In particular, the use of CID technology to generate self-assembled (and selectively disassembled) protein toroids serves as a key advancement toward developing stable and controllable protein-based platforms. Such structures have broad application to the development of novel therapeutics, lab-on-a-chip technologies, and multi-enzyme assemblies (5, 6). This chapter describes a method of developing an enzymatically active protein nanostructure incorporating both a CID-based assembly region containing dihydrofolate reductase (DHFR) and an enzymatic region consisting of histidine triad nucleotide binding protein 1 (Hint1). Details of both the production and the characterization of this structure are provided.

Methods in molecular biology (Clifton, N.J.), 2017

In recent years there has been increasing interest in nanostructure design based on the self-assembly properties of proteins and polymers. Nanodesign requires the ability to predictably manipulate the properties of the self-assembly of autonomous building blocks, which can fold or aggregate into preferred conformational states. The design includes functional synthetic materials and biological macromolecules. Autonomous biological building blocks with available 3D structures provide an extremely rich and useful resource. Structural databases contain large libraries of protein molecules and their building blocks with a range of sizes, shapes, surfaces, and chemical properties. The introduction of engineered synthetic residues or short peptides into these building blocks can greatly expand the available chemical space and enhance the desired properties. Herein, we summarize a protocol for designing nanostructures consisting of self-assembling building blocks, based on our recent works....

Nature communications, 2016

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate...

Journal of the American Chemical Society, 2012

The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide-and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represents a new route for constructing complex peptide-based assemblies and biomaterials.

2008

Directed, biologically-driven self-assembly has the potential to yield hybrid multicomponent architectures with applications ranging from sensors and diagnostics to nanoelectronic devices. Critical to these applications is to gain control over the precise orientation and geometry of biomolecules interacting with one-another and with surfaces. Such control has thus far been difficult to achieve in even the simplest biomolecular designs. Using a novel strategy for generation of multicomponent biological nanoarchitectures, the DNA-templated assembly of multiprotein complexes recognizing methylated DNA was achieved. The reassembly of two fragments of TEM-1 βlactamase, each one fused with a specific DNA recognition factor, into a catalytically active protein was achieved by using the cognate DNA elements of these factors. This strategy could potentially become a useful tool in studies of genomic DNA methylation in the context of cellular epigenetic processes.

Microbial Cell Factories, 2010

Like natural viruses, manmade protein cages for drug delivery are to be ideally formed by repetitive subunits with self-assembling properties, mimicking viral functions and molecular organization. Naturally formed nanostructures (such as viruses, flagella or simpler protein oligomers) can be engineered to acquire specific traits of interest in biomedicine, for instance through the addition of cell targeting agents for desired biodistribution and specific delivery of associated drugs. However, fully artificial constructs would be highly desirable regarding finest tuning and adaptation to precise therapeutic purposes. Although engineering of protein assembling is still in its infancy, arising principles and promising strategies of protein manipulation point out the rational construction of nanoscale protein cages as a feasible concept, reachable through conventional recombinant DNA technologies and microbial protein production.

2006

International Journal of Nanotechnology, 2010

Biologically programmed molecular recognition provides the basis of all natural systems and supplies evolution optimized functional materials from self-assembly of a limited number of molecular building blocks. Biomolecules such as peptides, nucleic acids and carbohydrates represent a diverse supply of structural building blocks for the chemist to design and fabricate new functional nanostructured architectures. In this context, we review here

Protein Science, 2013

The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(1) and A-(2), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C 3 symmetry. The coiledcoil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(1) and A-(2) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(1) and A-(2) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.