Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm.

We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.

Beyond icosahedral symmetry in packings of proteins in spherical shells.

The formation of quasi-spherical cages from protein building blocks is a remarkable self-assembly process in many natural systems, where a small number of elementary building blocks are assembled to build a highly symmetric icosahedral cage. In turn, this has inspired synthetic biologists to design de novo protein cages. We use simple models, on multiple scales, to investigate the self-assembly of a spherical cage, focusing on the regularity of the packing of protein-like objects on the surface. Using building blocks, which are able to pack with icosahedral symmetry, we examine how stable these highly symmetric structures are to perturbations that may arise from the interplay between flexibility of the interacting blocks and entropic effects. We find that, in the presence of those perturbations, icosahedral packing is not the most stable arrangement for a wide range of parameters; rather disordered structures are found to be the most stable. Our results suggest that (i) many designed, or even natural, protein cages may not be regular in the presence of those perturbations and (ii) optimizing those flexibilities can be a possible design strategy to obtain regular synthetic cages with full control over their surface properties.

Differential sensing with arrays of de novo designed peptide assemblies.

Differential sensing attempts to mimic the mammalian senses of smell and taste to identify analytes and complex mixtures. In place of hundreds of complex, membrane-bound G-protein coupled receptors, differential sensors employ arrays of small molecules. Here we show that arrays of computationally designed de novo peptides provide alternative synthetic receptors for differential sensing. We use self-assembling α-helical barrels (αHBs) with central channels that can be altered predictably to vary their sizes, shapes and chemistries. The channels accommodate environment-sensitive dyes that fluoresce upon binding. Challenging arrays of dye-loaded barrels with analytes causes differential fluorophore displacement. The resulting fluorimetric fingerprints are used to train machine-learning models that relate the patterns to the analytes. We show that this system discriminates between a range of biomolecules, drink, and diagnostically relevant biological samples. As αHBs are robust and chemically diverse, the system has potential to sense many analytes in various settings.

Designed coiled coils promote folding of a recombinant bacterial collagen.

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

De novo design of a reversible phosphorylation-dependent switch for membrane targeting.

Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a 'cargo' molecule reversibly to a permanent membrane 'anchor'; and (ii) creating a 'membrane-avidity switch' that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

Interactions of HIV-1 antibodies 2F5 and 4E10 with a gp41 epitope prebound to host and viral membrane model systems.

Two HIV-1 recognition domains for the human monoclonal antibodies (MAb) 2F5, which recognises the core sequence ELDKWA, and 4E10, which recognises the core sequence NWFNIT, serve as promising models for immunogens in vaccine development against HIV-1. However, the failure of these recognition domains to generate broadly reactive neutralizing antibodies, and the putative membrane-binding properties of the antibodies raised to these recognition domains, suggest that additional features or recognition motifs are required to form an efficient immunogen, which could possibly include the membrane components. In this study we used an extended peptide epitope sequence derived from the gp41 native sequence (H-NEQELLELDKWASLWNWFNITNWLWYIK-NH), which contains the two recognition domains for 2F5 and 4E10, to examine the role of model cell (POPC) and viral (POPC/cholesterol/sphingomyelin) membranes in the recognition of these two antibodies. By using a surface plasmon resonance biosensor, the binding of 2F5 and 4E10 to membranes was compared and contrasted in the presence and absence of prebound peptide epitope. The recognition of the peptide epitope by each MAb was found to be distinct; 2F5 exhibited strong and almost irreversible binding to both membranes in the presence of the peptide, but bound weakly in the absence of the peptide epitope. In contrast, 4E10 exhibited strong membrane binding in the presence or absence of the peptide epitope, and the binding was essentially irreversible in the presence of the peptide epitope. Overall, these results demonstrate that both 2F5 and 4E10 can bind to membranes prior to epitope recognition, but that high-affinity recognition of gp41-derived epitope sequences by 2F5 and 4E10 occurs in a membrane context. Moreover, 4E10 might utilise the membrane to access and bind to gp41; such membrane properties of 2F5 and 4E10 could be exploited in immunogen design.

Modified low molecular weight cyclic peptides as mimetics of BDNF with improved potency, proteolytic stability and transmembrane passage in vitro.

We recently reported the development of the BDNF mimetic peptide cyclo-[dPAKKR] 1 which promotes the survival of cultured sensory neurons via a trkB independent mechanism [Fletcher, J. M.; Morton, C. M.; Zwar, R. A.; Murray, S. S.; O'Leary, P. D.; Hughes, R. A. J. Biol. Chem.2008, 283, 33375]. In the present study we prepared a series of hydrophobically-modified analogues of 1 with an eye to improving its pharmacokinetic properties. Select members of this second generation of compounds showed improved biological activity, stability in plasma, and an ability to cross model biological membranes.

De novo coiled-coil peptides as scaffolds for disrupting protein-protein interactions.

Protein-protein interactions (PPIs) play pivotal roles in the majority of biological processes. Therefore, improved approaches to target and disrupt PPIs would provide tools for chemical biology and leads for therapeutic development. PPIs with α-helical components are appealing targets given that the secondary structure is well understood and can be mimicked or stabilised to render small-molecule and constrained-peptide-based inhibitors. Here we present a strategy to target α-helix-mediated PPIs that exploits de novo coiled-coil assemblies and test this using the MCL-1/NOXA-B PPI. First, computational alanine scanning is used to identify key α-helical residues from NOXA-B that contribute to the interface. Next, these residues are grafted onto the exposed surfaces of de novo designed homodimeric or heterodimeric coiled-coil peptides. The resulting synthetic peptides selectively inhibit a cognate MCL-1/BID complex in the mid-nM range. Furthermore, the heterodimeric system affords control as inhibition occurs only when both the grafted peptide and its designed partner are present. This establishes proof of concept for exploiting peptides stabilised in de novo coiled coils as inhibitors of PPIs. This dependence on supramolecular assembly introduces new possibilities for regulation and control.

De novo targeting to the cytoplasmic and luminal side of bacterial microcompartments.

Bacterial microcompartments, BMCs, are proteinaceous organelles that encase a specific metabolic pathway within a semi-permeable protein shell. Short encapsulation peptides can direct cargo proteins to the lumen of the compartments. However, the fusion of such peptides to non-native proteins does not guarantee encapsulation and often causes aggregation. Here, we report an approach for targeting recombinant proteins to BMCs that utilizes specific de novo coiled-coil protein-protein interactions. Attachment of one coiled-coil module to PduA (a component of the BMC shell) allows targeting of a fluorescent protein fused to a cognate coiled-coil partner. This interaction takes place on the outer surface of the BMC. The redesign of PduA to generate an N-terminus on the luminal side of the BMC results in intact compartments to which proteins can still be targeted via the designed coiled-coil system. This study provides a strategy to display proteins on the surface or within the lumen of the BMCs.

Bioinspired Silicification Reveals Structural Detail in Self-Assembled Peptide Cages.

Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g., staining or embedding, which can damage or obscure features. An alternative is to use bioinspired mineralization, mimicking how certain organisms use biomolecules to template mineral formation. Previously, we have reported the design and characterization of Self-Assembled peptide caGEs (SAGEs) formed from de novo peptide building blocks. In SAGEs, two complementary, 3-fold symmetric, peptide hubs combine to form a hexagonal lattice, which curves and closes to form SAGE nanoparticles. As hexagons alone cannot tile onto spheres, the network must also incorporate nonhexagonal shapes. While the hexagonal ultrastructure of the SAGEs has been imaged, these defects have not been observed. Here, we show that positively charged SAGEs biotemplate a thin, protective silica coating. Electron microscopy shows that these SiO2-SAGEs do not collapse, but maintain their 3D shape when dried. Atomic force microscopy reveals a network of hexagonal and irregular features on the SiO2-SAGE surface. The dimensions of these (7.2 nm ± 1.4 nm across, internal angles 119.8° ± 26.1°) are in accord with the designed SAGE network and with coarse-grained modeling of the SAGE assembly. The SiO2-SAGEs are permeable to small molecules (<2 nm), but not to larger biomolecules (>6 nm). Thus, bioinspired silicification offers a mild technique that preserves soft-matter nanoparticles for imaging, revealing structural details <10 nm in size, while also maintaining desirable properties, such as permeability to small molecules.

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces.

The α-helical coiled coil is one of the best-studied protein-protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix-helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.

Decorating Self-Assembled Peptide Cages with Proteins.

An ability to organize and encapsulate multiple active proteins into defined objects and spaces at the nanoscale has potential applications in biotechnology, nanotechnology, and synthetic biology. Previously, we have described the design, assembly, and characterization of peptide-based self-assembled cages (SAGEs). These ≈100 nm particles comprise thousands of copies of de novo designed peptide-based hubs that array into a hexagonal network and close to give caged structures. Here, we show that, when fused to the designed peptides, various natural proteins can be co-assembled into SAGE particles. We call these constructs pSAGE for protein-SAGE. These particles tolerate the incorporation of multiple copies of folded proteins fused to either the N or the C termini of the hubs, which modeling indicates form the external and internal surfaces of the particles, respectively. Up to 15% of the hubs can be functionalized without compromising the integrity of the pSAGEs. This corresponds to hundreds of copies giving mM local concentrations of protein in the particles. Moreover, and illustrating the modularity of the SAGE system, we show that multiple different proteins can be assembled simultaneously into the same particle. As the peptide-protein fusions are made via recombinant expression of synthetic genes, we envisage that pSAGE systems could be developed modularly to actively encapsulate or to present a wide variety of functional proteins, allowing them to be developed as nanoreactors through the immobilization of enzyme cascades or as vehicles for presenting whole antigenic proteins as synthetic vaccine platforms.

Self-assembling cages from coiled-coil peptide modules.

An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back to back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.

A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology.

Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.

Bioorthogonal dual functionalization of self-assembling peptide fibers.

The ability to modify peptide- and protein-based biomaterials selectively under mild conditions and in aqueous buffers is essential to the development of certain areas of bionanotechnology, tissue engineering and synthetic biology. Here we show that Self-Assembling peptide Fibers (SAFs) can incorporate multiple modified peptides non-covalently, stoichiometrically and without disrupting their structure or stability. The modified peptides contain groups suitable for post-assembly click reactions in water, namely azides and alkenes. Labeling of these groups is achieved using the orthogonal Cu(I)-catalyzed azide-alkyne and photoinitiated thiol-ene reactions, respectively. Functionalization is demonstrated through the conjugation of biotin followed by streptavidin-nanogold particles, or rhodamine, and visualized by electron and light microscopy, respectively. This has been shown for fibers harboring either or both of the modified peptides. Furthermore, the amounts of each modified peptide in the fibers can be varied with concomitant changes in decoration. This approach allows the design and assembly of fibers with multiple functional components, paving the way for the development of multi-component functionalized systems.

Synthesis of stapled beta3-peptides through ring-closing metathesis.

The first synthesis of carbon-stapled beta(3)-peptides is reported. The precursor beta(3)-peptides, with O-allyl beta-serines located in an i/i+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new beta(3)-peptides proceeds smoothly either in solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.

Design of a conformationally defined and proteolytically stable circular mimetic of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of neurotrophic factors. BDNF has long been recognized to have potential for the treatment of a variety of human neurodegenerative diseases. However, clinical trials with recombinant BDNF have yet to yield success, leading to the suggestion that alternative means of harnessing BDNF actions for therapeutic use may be required. Here we describe an approach to create low molecular weight peptides that, like BDNF, promote neuronal survival. The peptides were designed to mimic a cationic tripeptide sequence in loop 4 of BDNF shown in previous studies to contribute to the binding of BDNF to the common neurotrophin receptor p75NTR. The best of these peptides, the cyclic pentapeptide 2 (cyclo(-D-Pro-Ala-Lys-Arg-)), despite being of low molecular weight (Mr 580), was found to be an effective promoter of the survival of embryonic chick dorsal root ganglion sensory neurons in vitro (maximal survival, 68 +/- 3% of neurons supported by BDNF). Pentapeptide 2 did not affect the phosphorylation of either TrkB (the receptor tyrosine kinase for BDNF) or the downstream signaling molecule MAPK, indicating that its mechanism of neuronal survival action is independent of TrkB. NMR studies reveal that pentapeptide 2 adopts a well defined backbone conformation in solution. Furthermore, pentapeptide 2 was found to be effectively resistant to proteolysis when incubated in a solution of rat plasma in vitro. These properties of pentapeptide 2 (low molecular weight, appropriate pharmacological actions, a well defined solution conformation, and proteolytic stability) render it worthy of further investigation, either as a template for the further design of neuronal survival promoting agents or as a lead compound with therapeutic potential in its own right.

Novel monocyclic and bicyclic loop mimetics of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a protein that promotes the survival of neurons. It is widely thought to possess clinical potential for the treatment of neurodegenerative diseases, and in recent years, has been found to play a role in the pathogenesis of some tumours. BDNF is thought to bind to its cellular receptors trkB and p75(NTR) primarily by way of solvent-exposed loops on the BDNF dimer. In this paper, we describe our recent progress towards the development of small peptides as mimetics and inhibitors of BDNF. Two classes of peptides were prepared: disulphide-constrained monomeric monocyclic peptides designed to mimic a single solvent-exposed loop; and homo- and heterodimeric bicyclic peptides designed to mimic pairs of loops. Each peptide was examined in cultures of embryonic chick dorsal root ganglion sensory neurons, both alone, and in competition with BDNF. All peptides were found to inhibit BDNF-mediated neuronal survival, while one--a dimeric peptide based on the two loop 4 regions of BDNF--behaved as a partial BDNF-like agonist. The work described in this paper supports the proposed receptor-binding role of loops 1, 2, and 4 of BDNF, and provides valuable steps towards our long-term goal of developing BDNF mimetics and inhibitors for clinical use.