Orchestrating Chemical and Physical Cross-Linking in Protein Hydrogels to Regulate Embryonic Stem Cell Growth

Protein hydrogels are ideal candidates for next-generation biomaterials due to their genetically programmable properties. Herein, we report an entirely protein-based hydrogel as an artificial extracellular matrix (ECM) for regulating the embryonic stem cell growth. A synergy between chemical and physical cross-linking was achieved in one step by SpyTag/SpyCatcher reaction and P zipper association at 37 °C. The hydrogels’ stress relaxation behaviors can be tuned across a broad spectrum by single-point mutation on a P zipper. It has been found that faster relaxation can promote the growth of HeLa tumor spheroids and embryonic stem cells, and mechanical regulation of embryonic stem cells occurs via retention of the cells at the G1 phase. The results highlight the promise of genetically encoded protein materials as a platform of artificial ECM for understanding and controlling the complex cell–matrix interactions in a 3D cell culture

Controlling Macromolecular Topology with Genetically Encoded SpyTag–SpyCatcher Chemistry

Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag–SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification <i>in situ</i>. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 °C with 10 μM IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing <i>in vitro</i>. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag–SpyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag–SpyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure

B₁₂-Dependent Protein Oligomerization Facilitates Layer-by-Layer Growth of Photo/Thermal Responsive Nanofilms

We report the robust growth of an entirely protein-based, photo- and thermoresponsive Layer-by-Layer nanofilm using genetically encoded SpyTag/SpyCatcher chemistry. The process was facilitated by AdoB₁₂-induced tetramerization of photoreceptor proteins. Protein cargos can be released from the film in a light-dependent manner, showing its potential for therapeutic protein delivery

Higher Order Protein Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo Stability

The chemical topology is a unique dimension for protein engineering, yet the topological diversity and architectural complexity of proteins remain largely untapped. Herein, we report the biosynthesis of complex topological proteins using a rationally engineered, cross-entwining peptide heterodimer motif derived from p53dim (an entangled homodimeric mutant of the tetramerization domain of the tumor suppressor protein p53). The incorporation of an electrostatic interaction at specific sites converts the p53dim homodimer motif into a pair of heterodimer motifs with high specificity for directing chain entanglement upon folding. Its combination with split-intein-mediated ligation and/or SpyTag/SpyCatcher chemistry facilitates the programmed synthesis of protein heterocatenane or [n]­catenanes in cells, leading to a general and modular approach to complex protein catenanes containing various proteins of interest. Concatenation enhances not only the target protein’s affinity but also the in vivo stability as shown by its prolonged circulation time in blood. As a proof of concept, artificial antibodies have been developed by embedding a human epidermal growth factor receptor 2-specific affibody onto the [n]­catenane scaffolds and shown to exhibit a higher affinity and a better pharmacokinetic profile than the wild-type affibody. These results suggest that topology engineering holds great promise in the development of therapeutic proteins

Star-Shaped Polycyclic Aromatics Based on Oligothiophene-Functionalized Truxene: Synthesis, Properties, and Facile Emissive Wavelength Tuning

A facile approach to soluble star-shaped oligothiophene-functionalized polycyclic aromatics based on truxene is developed in this Communication. The Suzuki coupling reactions afford the thiophene-containing polycyclic aromatics with long branches (about 2.1 nm length from the heart to the periphery) from truxene precursor with excellent yields. The unsubstituted α-positions of thiophene rings allow for efficient halogenation and for further functionalization. The investigation of proton NMR spectra indicates that the hexahexyl groups efficiently prevent the self-association through the arene−arene π-stacking. Chemical shifts belonging to methylene groups move more upfield than do those of methyl groups. These chemical shift values (about 0.5−0.6 ppm) are quite lower than those of normal methyl and methylene groups. We also prepare a dendritic hyperbranched polymer P1 through FeCl3 mediated oxidative polymerizations. The photophysical properties of all compounds possessing good symmetry are investigated by UV−vis and emission measurement

Star-Shaped Polycyclic Aromatics Based on Oligothiophene-Functionalized Truxene: Synthesis, Properties, and Facile Emissive Wavelength Tuning

A facile approach to soluble star-shaped oligothiophene-functionalized polycyclic aromatics based on truxene is developed in this Communication. The Suzuki coupling reactions afford the thiophene-containing polycyclic aromatics with long branches (about 2.1 nm length from the heart to the periphery) from truxene precursor with excellent yields. The unsubstituted α-positions of thiophene rings allow for efficient halogenation and for further functionalization. The investigation of proton NMR spectra indicates that the hexahexyl groups efficiently prevent the self-association through the arene−arene π-stacking. Chemical shifts belonging to methylene groups move more upfield than do those of methyl groups. These chemical shift values (about 0.5−0.6 ppm) are quite lower than those of normal methyl and methylene groups. We also prepare a dendritic hyperbranched polymer P1 through FeCl3 mediated oxidative polymerizations. The photophysical properties of all compounds possessing good symmetry are investigated by UV−vis and emission measurement

SpyCatcher‑N^TEV: A Circularly Permuted, Disordered SpyCatcher Variant for Less Trace Ligation

The SpyTag/SpyCatcher reaction has emerged as a powerful way for bioconjugation, but it leaves a folded complex in the product after the formation of the isopeptide bond. To vary the location of the reactive residue and reduce the size of the complex and its potential immunogenicity, we engineer two circularly permuted SpyCatcher variants, SpyCatcher-N and SpyCatcher-N^TEV, the latter of which possesses a TEV-recognition site for removal of the fragment containing the catalytic site. Surprisingly, both variants are found to be disordered in solution, yet still retain the ability to form an ordered complex upon reaction with SpyTag with second-order rate constants of ∼10 M^–1 s^–1. Cellular expression of a telechelic protein bearing SpyCatcher-N^TEV at the N-terminus and SpyTag at the C-terminus gives both cyclized and chain-extended products. Notably, the monomers exist almost exclusively in the cyclic form owing to its high reactivity in vivo. The fragment containing the catalytic site of SpyCatcher-N^TEV can then be removed by TEV digestion, giving a circular protein with minimal trace from the ligation reaction. The plasticity of SpyTag/SpyCatcher reactive pair has promised an ever-expanding toolbox of genetically encoded peptide–protein reaction with versatile features

Extended π-Conjugated Dendrimers Based on Truxene

The largest π-conjugated dendrimers containing up to nine 10,15-dihydro-5H-diindeno[1,2-α;1‘,2‘;-c]fluorene (truxene) moieties have been prepared with good yields by repetitive Friedel−Crafts acetylation and acid-promoted cyclotrimerization reactions. An alternative approach to the convergent synthesis of desired dendrimers has been developed, in which the core is generated “in-situ” by acid-promoted cyclotrimerizations of aryl methyl ketones. This proves valuable to afford large-size and precisely well-defined dendrimers in an accelerated dendrimer-growth strategy utilizing enlarged repeat units. The increasing amount of SiCl4 dramatically improves the yield of cyclotrimerization reactions. The introduction of hexahexyl groups onto C-5, C-10, C-15 positions of the truxene moiety greatly enhances the solubility of our compounds. Further investigation indicates that the torsion angle between the truxene segment and the benzene ring might play a key role in determining the photo properties of π-conjugated dendrimers

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide–Protein Chemistry Enabled Multilayer Construction

Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide–protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody, and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to biosignals, such as Ca²⁺, trifluoperazine, and nicotinamide adenine dinucleotide phosphate (NADPH), and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multilayer setup exhibits a synergistic enhancement of DHFR activity with increasing numbers of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience