A Structure-Based Assembly Screen of Protein Cage Libraries in Living Cells: Experimentally Repacking a Protein-Protein Interface To Recover Cage Formation in an Assembly-Frustrated Mutant.

Cage proteins, which assemble into often highly symmetric hollow nanoscale capsules, have great potential in applications as far reaching as drug delivery, hybrid nanomaterial engineering, and catalysis. In addition, they are promising model systems for understanding how cellular nanostructures are constructed through protein-protein interactions, and they are beginning to be used as scaffolds for synthetic biology approaches. Recently, there has been renewed interest in the engineering of protein cages, and in support of these strategies, we have recently described a fluorescence-based assay for protein cage assembly that is specific for certain oligomerization states and symmetry-related protein-protein interfaces. In this work, we expand this assay to living cells and a high-throughput assay for screening protein cage libraries using flow cytometry. As a proof of principle, we apply this technique to the screening of libraries of a double-alanine mutant of the mini-ferritin, DNA-binding protein from starved cells (Dps). This mutant, due to disruption of key protein-protein interactions, is unable to assemble into a cage. Randomization of residues surrounding the double mutation afforded a repacked interface and proteins with recovered cage formation, demonstrating the strength and utility of this approach.

The Crystal Structure of a Maxi/Mini-Ferritin Chimera Reveals Guiding Principles for the Assembly of Protein Cages.

Cage proteins assemble into nanoscale structures with large central cavities. They play roles, including those as virus capsids and chaperones, and have been applied to drug delivery and nanomaterials. Furthermore, protein cages have been used as model systems to understand and design protein quaternary structure. Ferritins are ubiquitous protein cages that manage iron homeostasis and oxidative damage. Two ferritin subfamilies have strongly similar tertiary structure yet distinct quaternary structure: maxi-ferritins normally assemble into 24-meric, octahedral cages with C-terminal E-helices centered around 4-fold symmetry axes, and mini-ferritins are 12-meric, tetrahedral cages with 3-fold axes defined by C-termini lacking E-domains. To understand the role E-domains play in ferritin quaternary structure, we previously designed a chimera of a maxi-ferritin E-domain fused to the C-terminus of a mini-ferritin. The chimera is a 12-mer cage midway in size between those of the maxi- and mini-ferritin. The research described herein sets out to understand (a) whether the increase in size over a typical mini-ferritin is due to a frozen state where the E-domain is flipped out of the cage and (b) whether the symmetrical preference of the E-domain in the maxi-ferritin (4-fold axis) overrules the C-terminal preference in the mini-ferritin (3-fold axis). With a 1.99 Å resolution crystal structure, we determined that the chimera assembles into a tetrahedral cage that can be nearly superimposed with the parent mini-ferritin, and that the E-domains are flipped external to the cage at the 3-fold symmetry axes.

Designability of Aromatic Interaction Networks at E. coli Bacterioferritin B-Type Channels.

The bacterioferritin from E. coli (BFR), a maxi-ferritin made of 24 subunits, has been utilized as a model to study the fundamentals of protein folding and self-assembly. Through structural and computational analyses, two amino acid residues at the B-site interface of BFR were chosen to investigate the role they play in the self-assembly of nano-cage formation, and the possibility of building aromatic interaction networks at B-type protein-protein interfaces. Three mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, native gel electrophoresis, and temperature-dependent circular dichroism spectroscopy. All of the mutants fold into α-helical structures and possess lowered thermostability. The double mutant D132W/N34W was 12 °C less stable than the wild type, and was also the only mutant for which cage-like nanostructures could not be detected in the dried, surface-immobilized conditions of transmission electron microscopy. Two mutants-N34W and D132W/N34W-only formed dimers in solution, while mutant D132W favored the 24-mer even more robustly than the wild type, suggesting that we were successful in designing proteins with enhanced assembly properties. This investigation into the structure of this important class of proteins could help to understand the self-assembly of proteins in general.

Modular peptides from the thermoplastic squid sucker ring teeth form amyloid-like cross-ß supramolecular networks.

The hard sucker ring teeth (SRT) from decapodiforme cephalopods, which are located inside the sucker cups lining the arms and tentacles of these species, have recently emerged as a unique model structure for biomimetic structural biopolymers. SRT are entirely composed of modular, block co-polymer-like proteins that self-assemble into a large supramolecular network. In order to unveil the molecular principles behind SRT's self-assembly and robustness, we describe a combinatorial screening assay that maps the molecular-scale interactions between the most abundant modular peptide blocks of suckerin proteins. By selecting prominent interaction hotspots from this assay, we identified four peptides that exhibited the strongest homo-peptidic interactions, and conducted further in-depth biophysical characterizations complemented by molecular dynamic (MD) simulations to investigate the nature of these interactions. Circular Dichroism (CD) revealed conformations that transitioned from semi-extended poly-proline II (PII) towards ß-sheet structure. The peptides spontaneously self-assembled into microfibers enriched with cross ß-structures, as evidenced by Fourier-Transform Infrared Spectroscopy (FTIR) and Congo red staining. Nuclear Magnetic Resonance (NMR) experiments identified the residues involved in the hydrogen-bonded network and demonstrated that these self-assembled ß-sheet-based fibers exhibit high protection factors that bear resemblance to amyloids. The high stability of the ß-sheet network and an amyloid-like model of fibril assembly were supported by MD simulations. The work sheds light on how Nature has evolved modular sequence design for the self-assembly of mechanically robust functional materials, and expands our biomolecular toolkit to prepare load-bearing biomaterials from protein-based block co-polymers and self-assembled peptides. STATEMENT OF SIGNIFICANCE: The sucker ring teeth (SRT) located on the arms and tentacles of cephalopods represent as a very promising protein-based biopolymer with the potential to rival silk in biomedical and engineering applications. SRT are made of modular, block co-polymer like proteins (suckerins), which assemble into a semicrystalline polymer reinforced by nano-confined ß-sheets, resulting in a supramolecular network with mechanical properties that match those of the strongest engineering polymers. In this study, we aimed to understand the molecular mechanisms behind SRT's self-assembly and robustness. The most abundant modular peptidic blocks of suckerin proteins were studied by various spectroscopic methods, which demonstrate that SRT peptides form amyloid-like cross-ß structures.

Design and Applications of Protein-Cage-Based Nanomaterials.

Materials science is beginning to focus on biotemplation, and in support of that trend, it is realized that protein cages-proteins that assemble from multiple monomers into architectures with hollow interiors-can instill a number of unique advantages to nanomaterials. In addition, the structural and functional plasticity of many protein-cage systems permits their engineering for specific applications. In this review, the most commonly used viral and non-viral protein cages, which exhibit a wide diversity of size, functionality, and chemical and thermal stabilities, are described. Moreover, how they have been exploited for nanomaterial and nanotechnology applications is summarized.

Mutagenesis study to disrupt electrostatic interactions on the twofold symmetry interface of Escherichia coli bacterioferritin.

Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified and characterized. All of the mutants fold into α-helical structures. Consistent with the computational prediction, they all show a lowered thermostability; double mutant D56R-E60H was found to be 16°C less stable than the wild type. Except for the mutant E60H, all the other mutations completely shut down the formation of protein cages to favour the dimer state in solution. The mutants, however, retain their ability to form cage-like nanostructures in the dried, surface immobilized conditions of transmission electron microscopy. Our findings confirm that even a single charge-inversion mutation at the 2-fold interface of BFR can affect the quaternary structure of its dimers and their ability to self-assemble into cage structures.

Computationally assisted engineering of protein cages.

A hybrid computational method incorporating topographic analysis of protein surfaces and free-energy calculations of protein-protein interactions in protein nanocages is described. This design strategy can be used to engineer protein cages for enhanced structural stability and assembly.

Detection of protein cage assembly with bisarsenic fluorescent probes.

We describe a method for the detection of specific protein-protein interactions in protein cages through the exploitation of designed binding sites for bisarsenic fluorescent probes. These sites are engineered to be protein-protein interface specific. We have adapted this method to ferritins; however, it could conceivably be applied to other protein cages. It is thought that this technique could be utilized in the thermodynamic and kinetic characterization of cage assembly mechanisms and in the high-throughput screening of protein cage libraries for the discovery of proteins with new assembly properties or of optimized conditions for assembly.

Detection of specific protein-protein interactions in nanocages by engineering bipartite FlAsH binding sites.

Proteins that form cage-like structures have been of much recent cross-disciplinary interest due to their application to bioconjugate and materials chemistry, their biological functions spanning multiple essential cellular processes, and their complex structure, often defined by highly symmetric protein­protein interactions. Thus, establishing the fundamentals of their formation, through detecting and quantifying important protein­protein interactions, could be crucial to understanding essential cellular machinery, and for further development of protein-based technologies. Herein we describe a method to monitor the assembly of protein cages by detecting specific, oligomerization state dependent, protein­protein interactions. Our strategy relies on engineering protein monomers to include cysteine pairs that are presented proximally if the cage state assembles. These assembled pairs of cysteines act as binding sites for the fluorescent reagent FlAsH, which, once bound, provides a readout for successful oligomerization. As a proof of principle, we applied this technique to the iron storage protein, DNA-binding protein from starved cells from E. coli. Several linker lengths and conformations for the presentation of the cysteine pairs were screened to optimize the engineered binding sites. We confirmed that our designs were successful in both lysates and with purified proteins, and that FlAsH binding was dependent upon cage assembly. Following successful characterization of the assay, its throughput was expanded. A two-dimension matrix of pH and denaturing buffer conditions was screened to optimize nanocage stability. We intend to use this method for the high throughput screening of protein cage libraries and of conditions for the generation of inorganic nanoparticles within the cavity of these and other cage proteins.

Complete shift of ferritin oligomerization toward nanocage assembly via engineered protein-protein interactions.

Computational redesign of a dimorphic protein nano-cage at the C3-symmetrical interfaces forces it to assemble into the monomorphic cage. These monodisperse assemblies are at least 20 °C more stable than the parent. This approach adds to the toolkit of bottom-up molecular design with applications in protein engineering and hybrid nano-materials.

Rational disruption of the oligomerization of the mini-ferritin E. coli DPS through protein-protein interface mutation.

DNA-binding protein from starved cells (DPS), a mini-ferritin capable of self-assembling into a 12-meric nano-cage, was chosen as the basis for an alanine-shaving mutagenesis study to investigate the importance of key amino acid residues, located at symmetry-related protein-protein interfaces, in controlling protein stability and self-assembly. Nine mutants were designed through simple inspection, synthesized, and subjected to transmission electron microscopy, circular dichroism, size exclusion chromatography, and "virtual alanine scanning" computational analysis. The data indicate that many of these residues may be hot spot residues. Most remarkably, two residues, R83 and R133, were observed to shift the oligomerization state to ~50% dimer. Based on the hypothesis that these two residues constitute a "hot strip," located at the ferritin-like threefold axis, the double mutant was generated which completely shuts down detectable formation of 12-mer in solution, favoring a cooperatively folded dimer. The fact that this effect logically builds upon the single mutants emphasizes that complex self-assembly has the potential to be manipulated rationally. This study should have an impact on the fundamental understanding of the assembly of DPS protein cages specifically and protein quaternary structure in general. In addition, as there is much interest in applying these and similar systems to the templation of nano-materials and drug delivery, the ability to control this ferritin's oligomerization state and stability could prove especially valuable.

Self-assembly in the ferritin nano-cage protein superfamily.

Protein self-assembly, through specific, high affinity, and geometrically constraining protein-protein interactions, can control and lead to complex cellular nano-structures. Establishing an understanding of the underlying principles that govern protein self-assembly is not only essential to appreciate the fundamental biological functions of these structures, but could also provide a basis for their enhancement for nano-material applications. The ferritins are a superfamily of well studied proteins that self-assemble into hollow cage-like structures which are ubiquitously found in both prokaryotes and eukaryotes. Structural studies have revealed that many members of the ferritin family can self-assemble into nano-cages of two types. Maxi-ferritins form hollow spheres with octahedral symmetry composed of twenty-four monomers. Mini-ferritins, on the other hand, are tetrahedrally symmetric, hollow assemblies composed of twelve monomers. This review will focus on the structure of members of the ferritin superfamily, the mechanism of ferritin self-assembly and the structure-function relations of these proteins.

Stabilization of a protein nanocage through the plugging of a protein-protein interfacial water pocket.

The unique structural properties of the ferritin protein cages have provided impetus to focus on the methodical study of these self-assembling nanosystems. Among these proteins, Escherichia coli bacterioferritin (EcBfr), although architecturally very similar to other members of the family, shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states. Through computational analysis and comparison to its homologues, we have found that this protein has a smaller than average dimeric interface on its 2-fold symmetry axis mainly because of the existence of an interfacial water pocket centered around two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, we have used a semiempirical computational method to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to that of wild-type EcBfr. This computational study also converged on the water-bridged asparagines. Replacing these two asparagines with hydrophobic amino acids resulted in proteins that folded into α-helical monomers and assembled into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirmed that, in all cases, these proteins, in agreement with the calculations, possessed increased stability. One of the three mutations shifts the population in favor of the higher-order oligomerization state in solution as evidenced by both size exclusion chromatography and native gel electrophoresis. These results taken together suggest that our low-level design was successful and that it may be possible to apply the strategy of targeting water pockets at protein--protein interfaces to other protein cage and self-assembling systems. More generally, this study further demonstrates the power of jointly employing in silico and in vitro techniques to understand and enhance biostructural energetics.

The discovery of small-molecule mimicking peptides through phage display.

Using peptides to achieve the functional and structural mimicry of small-molecules, especially those with biological activity or clear biotechnological applications, has great potential in overcoming difficulties associated with synthesis, or unfavorable physical properties. Combinatorial techniques like phage display can aid in the discovery of these peptides even if their mechanism of mimicry is not rationally obvious.The major focus of this field has been limited to developing biotin and sugar mimetics. However, the full "mimicry" of these peptides has not yet been fully established as some bind to the target with a different mechanism than that of the natural ligand and some do not share all of the natural ligand's binding partners. In this article, mimicry of small-molecules by phage display-discovered peptides is reviewed and their potential in biochemical and medical applications is analyzed.

Peptide ligands that use a novel binding site to target both TGF-ß receptors.

The transforming growth factor beta (TGF-ß) signaling pathway plays myriad roles in development and disease. TGF-ß isoforms initiate signaling by organizing their cell surface receptors TßRI and TßRII. Exploration and exploitation of the versatility of TGF-ß signaling requires an enhanced understanding of structure-function relationships in this pathway. To this end, small molecule, peptide, and antibody effectors that bind key signaling components would serve as valuable probes. We focused on the extracellular domain of TßR1 (TßRI-ED) as a target for effector screening. The observation that TßRI-ED can bind to a TGF-ß coreceptor (endoglin) suggests that the TßRI-ED may have multiple interaction sites. Using phage display, we identified two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the TßRI-ED (K(d)≈ 10(-5) M). Although our screen focused on TßRI-ED, the hit peptides interact with the TßRII-ED with similar affinities. The peptide ligands occupy the same binding sites on TßRI and TßRII, as demonstrated by their ability to compete with each other for receptor binding. Moreover, neither interferes with TGF-ß binding. These results indicate that both TßRI and TßRII possess hot spots for protein-protein interactions that are distinct from those used by their known ligand TGF-ß. To convert these compounds into high affinity probes, we exploited the observation that TßRI and TßRII exist as dimers on the cell surface; therefore, we assembled a multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the role of receptor assembly in TGF-ß function.

Alanine-shaving mutagenesis to determine key interfacial residues governing the assembly of a nano-cage maxi-ferritin.

The fundamental process of protein self-assembly is governed by protein-protein interactions between subunits, which combine to form structures that are often on the nano-scale. The nano-cage protein, bacterioferritin from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for an alanine-shaving mutagenesis study to discover key amino acid residues at symmetry-related protein-protein interfaces that control protein stability and self-assembly. By inspection of these interfaces and "virtual alanine scanning," nine mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, dynamic light scattering, native PAGE, and temperature-dependent CD. Many of the selected amino acids act as hot spot residues. Four of these (Arg-30, which is located at the two-fold axis, and Arg-61, Tyr-114, and Glu-128, which are located at the three-fold axis), when individually mutated to alanine, completely shut down detectable solution formation of 24-mer, favoring a cooperatively folded dimer, suggesting that they may be oligomerization "switch residues." Furthermore, two residues, Arg-30 and Arg-61, when changed to alanine form mutants that are more thermodynamically stable than the native protein. This investigation into the structure and energetics of this self-assembling nano-cage protein not only can act as a jumping off point for the eventual design of novel protein nano-structures but can also help to understand the role that structure plays on the function of this important class of proteins.

High-throughput discovery of synthetic surfaces that support proliferation of pluripotent cells.

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors, the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors, however, may be needed to guide cell growth and differentiation. To provide access to such new materials, we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait, approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested, and seven cell-binding peptides were identified. Of these, six sequences possess EC cell-binding ability. Specifically, when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold, they mediate cell adhesion. The corresponding soluble peptides block this adhesion, indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR), they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells.

A helix swapping study of two protein cages.

Protein cages have been the focus of studies across multiple scientific disciplines. They have been used to deliver drugs, as templates for nanostructured materials, as substrates in the development of bio-orthogonal chemistry, and to restrict diffusion to study spatially confined reactions. Although their monomers fold into four-helix bundle structures, two cage proteins, DPS and BFR, self-assemble to form a 12-mer with tetrahedral symmetry and an octahedrally symmetric 24-mer, respectively. These monomers share strong similarities of both sequence and tertiary structure. However, they differ in the presence of a short additional helix. In BFR, the fifth helix is at the C-terminus and is positioned along the 4-fold symmetry axis, whereas with DPS, an extra helix helps to define the 2-fold axis in the cage and is located between the second and third helices in the monomer bundle. In an attempt to investigate if these short helices govern protein assembly, mutants were designed and produced that delete and swap these minidomains. All mutants form highly helical structures that unfold cooperatively as evidenced by thermal melting followed by circular dichroism. Dynamic light scattering, size exclusion chromatography, and sedimentation equilibrium experiments demonstrated that although many of the BFR mutants do not self-assemble and form lower-order complexes, many DPS mutants could form cages despite their unnatural design. Taken together, our data indicate that the BC helix is less important than the E helix for overall cage self-assembly, suggesting that dimerization may not play a role in nanostructure formation that is as key as previously assumed. Additionally, we found that fusing the minidomain from BFR onto DPS results in a mutant that assembles into a homogeneous population of a novel protein oligomer. This assembled cage while still formed from 12 subunits is larger in overall shape than that of the native protein.

Phage display screening against a set of targets to establish peptide-based sugar mimetics and molecular docking to predict binding site.

A novel selection approach is presented to screen phage display peptide libraries against sets of receptors that share specificity for the same ligand. This strategy was applied to the discovery of glycomimetic peptides. Through these screens, a number of peptide clones were discovered that bind the lectins used in the screen, in a sugar competitive manner. In addition, the majority of the selected peptides demonstrate sugar type mimicry consistent with lectin specificity. Docking studies were conducted to establish whether the mimetic peptides bind to the lectin ConA at the sugar binding site or to a nearby, alternative site shown to bind to YPY-containing peptides previously discovered from single-target screens. Of the three cyclic peptides subjected to computational docking, CNTPLTSRC had the highest predicted affinity and CSRILTAAC demonstrated specificity for the sugar binding site comparable to the natural ligand itself.