Structure by design: from single proteins and their building blocks to nanostructures

Nanotechnology realizes the advantages of naturally occurring biological macromolecules and their building-block nature for design. Frequently, assembly starts with the choice of a "good" molecule that is synthetically optimized towards the desired shape. By contrast, we propose starting with a pre-specified nanostructure shape, selecting candidate protein building blocks from a library and mapping them onto the shape and, finally, testing the stability of the construct. Such a shape-based, part-assembly strategy is conceptually similar to protein design through the combinatorial assembly of building blocks. If the conformational preferences of the building blocks are retained and their interactions are favorable, the nanostructure will be stable. The richness of the conformations, shapes and chemistries of the protein building blocks suggests a broad range of potential applications; at the same time, it also highlights their complexity. In this Opinion article, we focus on the first step: validating such a strategy against experimental data.     

Symmetry-based self-assembled nanotubes constructed using native protein structures: the key role of flexible linkers

We construct nanotubes using native protein structures and their native associations from structural databases. The construction is based on a shape-guided symmetric self-assembly concept. Our strategy involves fusing judiciously-selected oligomerization domains via peptide linkers. Linkers are inherently flexible, hence their choice is critical: they should position the domains in three-dimensional space in the desired orientation while retaining their own natural conformational tendencies; however, at the same time, retain the construct stability. Here we outline a design scheme which accounts for linker flexibility considerations, and present two examples. The first is HIV-1 capsid protein, which in vitro self-assembles into nanotubes and conical capsids, and its linker exists as a short flexible loop. The second involves novel nanotubes construction based on antimicrobial homodimer Magainin 2, employing linkers of distinct lengths and flexibility levels. Our strategy utilizes the abundance of unique shapes and sizes of proteins and their building blocks which can assemble into a vast number of combinations, and consequently, nanotubes of distinct morphologies and diameters. Computational design and assessment methodologies can help reduce the number of candidates for experimental validation. This is an invited paper for a special issue on protein dynamics, here focusing on flexibility in nanotube design based on protein building blocks.     

Biologically inspired strategy for programmed assembly of viral building blocks with controlled dimensions

Facile fabrication of building blocks with precisely controlled dimensions is imperative in the development of functional devices and materials. We demonstrate the assembly of nanoscale viral building blocks of controlled lengths using a biologically motivated strategy. To achieve this we exploit the simple self-assembly mechanism of Tobacco mosaic virus (TMV), whose length is solely governed by the length of its genomic mRNA. We synthesize viral mRNA of desired lengths using simple molecular biology techniques, and in vitro assemble the mRNA with viral coat proteins to yield viral building blocks of controlled lengths. The results indicate that the assembly of the viral building blocks is consistent and reproducible, and can be readily extended to assemble building blocks with genetically modified coat proteins (TMV1cys). Additionally, we confirm the potential utility of the TMV1cys viral building blocks with controlled dimensions via covalent and quantitative conjugation of fluorescent markers. We envision that our biologically inspired assembly strategy to design and construct viral building blocks of controlled dimensions could be employed to fabricate well-controlled nanoarchitectures and hybrid nanomaterials for a wide variety of applications including nanoelectronics and nanocatalysis.     

Principles of nanostructure design with protein building blocks

Currently there is increasing interest in nanostructures and their design. Nanostructure design involves the ability to predictably manipulate the properties of the self-assembly of autonomous units. Autonomous units have preferred conformational states. The units can be synthetic material science-based or derived from functional biological macromolecules. Autonomous biological building blocks with available structures provide an extremely rich and useful resource for design. For proteins, the structural databases contain large libraries of protein molecules and their building blocks with a range of shapes, surfaces, and chemical properties. The introduction of engineered synthetic residues or short peptides into these can expand the available chemical space and enhance the desired properties. Here we focus on the principles of nanostructure design with protein building blocks.     

Nanostructure design using protein building blocks enhanced by conformationally constrained synthetic residues

Increasing efforts are being invested in the construction of nanostructures with desired shapes and physical and chemical properties. Our strategy involves nanostructure design using naturally occurring protein building blocks. Inspection of the protein structural database (PDB) reveals the richness of the conformations, shapes, and chemistries of proteins and their building blocks. To increase the population of the native fold in the selected building block, we mutate natural residues by engineered, constrained residues that restrict the conformational freedom at the targeted site and have favorable interactions, geometry, and size. Here, as a model system, we construct nanotubes using building blocks from left-handed beta-helices which are commonly occurring repeat protein architectures. We pick two-turn beta-helical segments, duplicate and stack them, and using all-atom molecular dynamics simulations (MD) with explicit solvent probe the structural stability of these nanotubular structures as indicated by their capacity to retain the initial organization and their conformational dynamics. Comparison of the results for the wild-type and mutated sequences shows that the introduction of the conformationally restricted 1-aminocyclopropanecarboxylic acid (Ac3c) residue in loop regions greatly enhances the stability of beta-helix nanotubes. The Ac3c geometrical confinement effect is sequence-specific and position-specific. The achievement of high stability of nanotubular structures originates not only from the reduction of mobility at the mutation site induced by Ac3c but also from stabilizing association forces between building blocks such as hydrogen bonds and hydrophobic contacts. For the selected synthetic residue, similar size, hydrophobicity, and backbone conformational tendencies are desirable as in the Ac3c.     

In silico protein design by combinatorial assembly of protein building blocks

Utilizing concepts of protein building blocks, we propose a de novo computational algorithm that is similar to combinatorial shuffling experiments. Our goal is to engineer new naturally occurring folds with low homology to existing proteins. A selected protein is first partitioned into its building blocks based on their compactness, degree of isolation from the rest of the structure, and hydrophobicity. Next, the protein building blocks are substituted by fragments taken from other proteins with overall low sequence identity, but with a similar hydrophobic/hydrophilic pattern and a high structural similarity. These criteria ensure that the designed protein has a similar fold, low sequence identity, and a good hydrophobic core compared with its native counterpart. Here, we have selected two proteins for engineering, protein G B1 domain and ubiquitin. The two engineered proteins share approximately 20% and approximately 25% amino acid sequence identities with their native counterparts, respectively. The stabilities of the engineered proteins are tested by explicit water molecular dynamics simulations. The algorithm implements a strategy of designing a protein using relatively stable fragments, with a high population time. Here, we have selected the fragments by searching for local minima along the polypeptide chain using the protein building block model. Such an approach provides a new method for engineering new proteins with similar folds and low homology.     

Synthetic circuits,devices and modules

The aim of synthetic biology is to design artificial biological systems for novel applications. From an engineering perspective, construction of biological systems of defined functionality in a hierarchical way is fundamental to this emerging field. Here, we highlight some current advances on design of several basic building blocks in synthetic biology including the artificial gene control elements, synthetic circuits and their assemblies into devices and modules. Such engineered basic building blocks largely expand the synthetic toolbox and contribute to our understanding of the underlying design principles of living cells.     

Self-assembly of repeat proteins: Concepts and design of new interfaces

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein–protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose.In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.     

Use of RNA structure flexibility data in nanostructure modeling

In the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Our goal is to develop computer methods for aiding in this process. Towards that end, we created the RNA junction database, which is a repository of RNA junctions, i.e. internal, multi-branch and kissing loops with emanating stem stubs, extracted from the larger RNA structures stored in the PDB database. These junctions can be used as building blocks for nanostructures. Two programs developed in our laboratory, NanoTiler and RNA2D3D, can combine such building blocks with idealized fragments of A-form helices to produce desired 3D nanostructures. Initially, the building blocks are treated as rigid objects and the resulting geometry is tested against the design objectives. Experimental data, however, shows that RNA accommodates its shape to the constraints of larger structural contexts. Therefore we are adding analysis of the flexibility of our building blocks to the full design process. Here we present an example of RNA-based nanostructure design, putting emphasis on the need to characterize the structural flexibility of the building blocks to induce ring closure in the automated exploration. We focus on the use of kissing loops (KL) in nanostructure design, since they have been shown to play an important role in RNA self-assembly. By using an experimentally proven system, the RNA tectosquare, we show that considering the flexibility of the KLs as well as distortions of helical regions may be necessary to achieve a realistic design.     

Solid-phase synthesis of second-generation polyproline dendrimers

Second-generation dendrimers have been prepared on solid phase by successive additions of branched polyproline building blocks starting from two different branching units anchored to the solid support. The preparation of Pro-rich building blocks was carried out by stepwise solid-phase synthesis and their iterative addition was performed by a convergent approach, also using solid-phase synthesis. cis-4-Amino-L-proline and imidazolidine-2-carboxylic acid were used as branching units due to their structural resemblance to proline. The optimized strategy allowed the target compounds to be obtained with high purities without the need for purification steps.     

A coarse-grained approach to protein design: learning from design to understand folding

Computational studies have given a great contribution in building our current understanding of the complex behavior of protein molecules; nevertheless, a complete characterization of their free energy landscape still represents a major challenge. Here, we introduce a new coarse-grained approach that allows for an extensive sampling of the conformational space of a large number of sequences. We explicitly discuss its application in protein design, and by studying four representative proteins, we show that the method generates sequences with a relatively smooth free energy surface directed towards the target structures.     

New reagents, reactions, and peptidomimetics for drug design.

It has been a major focus in our laboratories to prepare novel reagents and peptidomimetic structures for drug design. We have designed and prepared novel guanidinylation reagents that can be employed in solution or as solid phase reagents. We and others have utilized the reagent 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) for amide bond formation to couple sterically hindered structures. These couplings proceed with remarkably strong resistance to racemization. In the area of peptidomimetics, we have incorporated novel building blocks to create biologically active compounds. These building blocks include thioether and alkylamine bridges, beta-methylated, and beta,beta-dimethylated amino acid residues. These mimetic structures have been incorporated into specific target molecules such as opioids to obtain cyclic peptidomimetics with potent and selective biological activity.     

Structure-based ligand design by dynamically assembling molecular building blocks at binding site.

A structure-based ligand design method is proposed and tested. The method is based on stochastic dynamics simulation of multiple copies of molecular building blocks in the presence of a receptor molecule. The molecular building blocks are assembled into candidate compounds "on the fly" at given intervals during the simulation. In the algorithm, a special effort is made to explore different possible combinations of building blocks and to select an optimum combination. By repeating the cycle of deconstruction and reconstruction in a single simulation, a set of candidate compounds that can be built from the building blocks evolves and is dynamically optimized. The method was tested by breaking two known flexible human immunodeficiency virus type 1 protease inhibitors into building blocks and reassembling them in the active site of the enzyme. For the inhibitor L700417, a set of conformations was generated by the calculation. Among these, the original compound was recovered with the lowest energy at the experimentally observed binding site and in the correct conformation. For pepstatin, the experimentally observed binding mode of the backbone of the inhibitor was reproduced by a calculation in which the building blocks corresponding to the side-chain groups were omitted. Proteins 1999;36:462-470.     

Hierarchical protein folding pathways: a computational study of protein fragments

We have previously presented a building block folding model. The model postulates that protein folding is a hierarchical top-down process. The basic unit from which a fold is constructed, referred to as a hydrophobic folding unit, is the outcome of combinatorial assembly of a set of "building blocks." Results obtained by the computational cutting procedure yield fragments that are in agreement with those obtained experimentally by limited proteolysis. Here we show that as expected, proteins from the same family give very similar building blocks. However, different proteins can also give building blocks that are similar in structure. In such cases the building blocks differ in sequence, stability, contacts with other building blocks, and in their 3D locations in the protein structure. This result, which we have repeatedly observed in many cases, leads us to conclude that while a building block is influenced by its environment, nevertheless, it can be viewed as a stand-alone unit. For small-sized building blocks existing in multiple conformations, interactions with sister building blocks in the protein will increase the population time of the native conformer. With this conclusion in hand, it is possible to develop an algorithm that predicts the building block assignment of a protein sequence whose structure is unknown. Toward this goal, we have created sequentially nonredundant databases of building block sequences. A protein sequence can be aligned against these, in order to be matched to a set of potential building blocks.     

Testing beta-helix terminal coils stability by targeted substitutions with non-proteogenic amino acids: a molecular dynamics study

The search for new building block templates useful for nanostructures design, targets protein motifs with a wide range of structures. Stabilizing these building blocks when extracted from their natural environment becomes a fundamental goal in order to successfully control their assembly. Targeted replacements of natural residues by conformationally constrained amino acids were shown to be a successful strategy to achieve such stabilization. In this work, the effect of replacing natural amino acids by non-proteogenic residues in a beta-helix building block has been evaluated using extensive molecular dynamics simulations. Here, we focus on systematic substitutions of valine residues present in beta-sheet segments of a beta-helical building block excised from Escherichia coli galactoside acetyltransferase, residues 131-165. Four different types of non-proteogenic amino acids have been considered for substitution: (i) one dehydroamino acid, (ii) two d-amino acids, (iii) one beta-amino acid and (iv) two alpha,alpha-dialkylamino acids. Our results indicate that the ability of non-proteogenic amino acids to stabilize small building block motifs is site-dependent. We conclude that if the replacement does not alter the energy balance between attractive non-covalent interactions and steric hindrance, synthetic residues are suitable candidates to nucleate beta-helix formation.     

Stability of tubular structures based on beta-helical proteins: self-assembled versus polymerized nanoconstructs and wild-type versus mutated sequences

In this work we used atomistic molecular dynamics simulations to examine different aspects of tubular nanostructures constructed using protein building blocks with a beta-helical conformation. Initially, we considered two different natural protein building blocks, which were extracted from the protein data base, to compare the relative stabilities of the nanotubes obtained made of self-assembled and covalently linked repeats. Results show nanotubes constructed by linking building blocks through covalent bonds are very stable suggesting that the basic principles of polymer physics are valid when the repeating units are made of large fragments of proteins. In contrast, the stability of self-assembled nanostructures strongly depends on the attractive nonbonding interactions associated to building blocks aligned in a complementary manner. On the other hand, we investigated the ability of a conformationally constrained synthetic amino acid to enhance the stability of both self-assembled and polymerized nanotubes when it is used to substitute natural residues. Specifically, we considered 1-aminocyclopentane-1-caboxylic acid, which involves strong stereochemical constraints produced by the cyclopentane side chain. We found that the incorporation of this amino acid within the more flexible regions of the beta-helical building blocks is an excellent strategy to enhance the stability of the nanotubes. Thus, when a single mutation is performed in the loop region of the beta-helix, the bend architecture of the whole loop is stabilized since the conformational mobility is reduced not only at the mutated position but also at the adjacent positions.     

Solid-phase route to Fmoc-protected cationic amino acid building blocks

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(β)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.     

A 3D building blocks approach to analyzing and predicting structure of proteins

A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of C alpha coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set of standard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can "replace" about 76% of all hexamers in well-refined known proteins with an error of less than 1 A, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.     

Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2'-deoxyuridine moieties

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of DNA oligonucleotides that bear 5-(mercaptomethyl)-2'-deoxyuridine moieties. The building blocks were synthesized in a straightforward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the generation of modified oligonucleotides containing the thiolated building blocks.