Rapid Production of Native and Mirror-Image Tumor Necrosis Factor-α Enabled by Automated Flow Peptide Synthesis Technology.

Tumor necrosis factor-α (TNF-α) plays a central role in immune response regulation. Because elevated TNF-α production is correlated with a range of diseases, inhibiting the interaction of this protein with its native receptors has been thoroughly explored as a therapeutic avenue. Despite advancements in the development of TNF-α inhibitors, concerns remain regarding immunogenicity and loss of activity in vivo. To facilitate the discovery of stable and less immunogenic therapeutic modalities, we applied a single-shot automated fast-flow peptide synthesis (AFPS) strategy to produce full-length TNF-α, resulting in a complex reaction mixture. Leveraging the ability of AFPS to generate long peptides with high purity, we combined this technology with native chemical ligation (NCL). An NCL reaction using two fragments readily produced by AFPS afforded synthetic L- and D-TNF-α in milligram quantities (up to 5.5 mg, ∼28% yield). Following the oxidative folding of synthetic TNF-α using established conditions, higher molecular weight species were generated. Through high-throughput screening of refolding conditions, functional synthetic L- and mirror-image D-TNF-α were obtained, exhibiting characteristics analogous to those of the recombinant TNF-α. Overall, this approach can serve as a general protocol for accessing proteins that are intractable by modern protein synthesis methods, therefore, streamlining the development of novel therapeutics.

Mirror-image ligand discovery enabled by single-shot fast-flow synthesis of D-proteins.

Widespread adoption of mirror-image biological systems presents difficulties in accessing the requisite D-protein substrates. In particular, mirror-image phage display has the potential for high-throughput generation of biologically stable macrocyclic D-peptide binders with potentially unique recognition modes but is hindered by the individualized optimization required for D-protein chemical synthesis. We demonstrate a general mirror-image phage display pipeline that utilizes automated flow peptide synthesis to prepare D-proteins in a single run. With this approach, we prepare and characterize 12 D-proteins - almost one third of all reported D-proteins to date. With access to mirror-image protein targets, we describe the successful discovery of six macrocyclic D-peptide binders: three to the oncoprotein MDM2, and three to the E3 ubiquitin ligase CHIP. Reliable production of mirror-image proteins can unlock the full potential of D-peptide drug discovery and streamline the study of mirror-image biology more broadly.

Recognition and reprogramming of E3 ubiquitin ligase surfaces by α-helical peptides.

Molecules that induce novel interactions between proteins hold great promise for the study of biological systems and the development of therapeutics, but their discovery has been limited by the complexities of rationally designing interactions between three components, and because known binders to each protein are typically required to inform initial designs. Here, we report a general and rapid method for discovering α-helically constrained (Helicon) polypeptides that cooperatively induce the interaction between two target proteins without relying on previously known binders or an intrinsic affinity between the proteins. We show that Helicons are capable of binding every major class of E3 ubiquitin ligases, which are of great biological and therapeutic interest but remain largely intractable to targeting by small molecules. We then describe a phage-based screening method for discovering "trimerizer" Helicons, and apply it to reprogram E3s to cooperatively bind an enzyme (PPIA), a transcription factor (TEAD4), and a transcriptional coactivator (ß-catenin).

De novo mapping of α-helix recognition sites on protein surfaces using unbiased libraries.

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Effect of Terminal Modification on the Molecular Assembly and Mechanical Properties of Protein-Based Block Copolymers.

Accurate prediction and validation of the assembly of bioinspired peptide sequences into fibers with defined mechanical characteristics would aid significantly in designing and creating materials with desired properties. This process may also be utilized to provide insight into how the molecular architecture of many natural protein fibers is assembled. In this work, computational modeling and experimentation are used in tandem to determine how peptide terminal modification affects a fiber-forming core domain. Modeling shows that increased terminal molecular weight and hydrophilicity improve peptide chain alignment under shearing conditions and promote consolidation of semicrystalline domains. Mechanical analysis shows acute improvements to strength and elasticity, but significantly reduced extensibility and overall toughness. These results highlight an important entropic function that terminal domains of fiber-forming peptides exhibit as chain alignment promoters, which ultimately has notable consequences on the mechanical behavior of the final fiber products.

Predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres.

Scalable computational modelling tools are required to guide the rational design of complex hierarchical materials with predictable functions. Here, we utilize mesoscopic modelling, integrated with genetic block copolymer synthesis and bioinspired spinning process, to demonstrate de novo materials design that incorporates chemistry, processing and material characterization. We find that intermediate hydrophobic/hydrophilic block ratios observed in natural spider silks and longer chain lengths lead to outstanding silk fibre formation. This design by nature is based on the optimal combination of protein solubility, self-assembled aggregate size and polymer network topology. The original homogeneous network structure becomes heterogeneous after spinning, enhancing the anisotropic network connectivity along the shear flow direction. Extending beyond the classical polymer theory, with insights from the percolation network model, we illustrate the direct proportionality between network conductance and fibre Young's modulus. This integrated approach provides a general path towards de novo functional network materials with enhanced mechanical properties and beyond (optical, electrical or thermal) as we have experimentally verified.

Silk-Its Mysteries, How It Is Made, and How It Is Used.

This article reviews fundamental and applied aspects of silk-one of Nature's most intriguing materials in terms of its strength, toughness, and biological role-in its various forms, from protein molecules to webs and cocoons, in the context of mechanical and biological properties. A central question that will be explored is how the bridging of scales and the emergence of hierarchical structures are critical elements in achieving novel material properties, and how this knowledge can be explored in the design of synthetic materials. We review how the function of a material system at the macroscale can be derived from the interplay of fundamental molecular building blocks. Moreover, guidelines and approaches to current experimental and computational designs in the field of synthetic silklike materials are provided to assist the materials science community in engineering customized finetuned biomaterials for biomedical applications.

Multifunctional spider silk polymers for gene delivery to human mesenchymal stem cells.

Non-viral gene delivery systems are important transport vehicles that can be safe and effective alternatives to currently available viral systems. A new family of multifunctional spider silk-based gene carriers was bioengineered and found capable of targeting human mesenchymal stem cells (hMSCs). These carriers successfully delivered DNA to the nucleus of these mammalian cells. The presence of specific functional sequences in the recombinant proteins, such as a nuclear localization sequence (NLS) of the large tumor (T) antigen of the Simian virus 40 (SV40 ), an hMSC high affinity binding peptide (HAB), and a translocation motif (TLM) of the hepatitis-B virus surface protein (PreS2), and their roles in mitigation and enhancement of gene transfection efficiency towards hMSCs were characterized. The results demonstrate that these bioengineered spider silk proteins serve as effective carriers, without the well-known complications associated with viral delivery systems.

Bioengineered silk gene delivery system for nuclear targeting.

Gene delivery research has gained momentum with the use of lipophilic vectors that mimic viral systems to increase transfection efficiency. Maintaining cell viability with these systems remains a major challenge. Therefore, biocompatible biopolymers that are designed by combining non-immunological viral mimicking components with suitable carrier are explored to address these limitations. In the present study, dragline silk recombinant proteins are modified with DNA condensing units and the proton sponge endosomal escape pathway is utilized for enhanced delivery. Transfection efficiency in a COS-7 cell line is enhanced compared to lipofectamine and polyethyleneimine (PEI), as is cell viability.

Effect of sequence features on assembly of spider silk block copolymers.

Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions.

Structure-function-property-design interplay in biopolymers: spider silk.

Spider silks have been a focus of research for almost two decades due to their outstanding mechanical and biophysical properties. Recent advances in genetic engineering have led to the synthesis of recombinant spider silks, thus helping to unravel a fundamental understanding of structure-function-property relationships. The relationships between molecular composition, secondary structures and mechanical properties found in different types of spider silks are described, along with a discussion of artificial spinning of these proteins and their bioapplications, including the role of silks in biomineralization and fabrication of biomaterials with controlled properties.

Stability of silk and collagen protein materials in space.

Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered.

Recombinant DNA production of spider silk proteins.

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.