Dynamically Tunable, Macroscopic Molecular Networks Enabled by Cellular Synthesis of 4-Arm Star-like Proteins

Abstract

Design of proteins with nonlinear topologies has emerged as a nascent branch of protein engineering, but significant applications remain to be seen. Here, we demonstrate the cellular synthesis of (SpyCatcher) 4 GFP, a 4-arm star-like protein enabled by spontaneous split GFP reconstitution, which further led to the creation of various protein networks exhibiting tunable mechanics and suitability for cell encapsulation. A derivative 4-arm star-like protein, (CarH C ) 4 GFP, resulting from the conjugation of (SpyCatcher) 4 GFP with the SpyTag-fusion CarH C photoreceptors, can undergo rapid sol-gel and gel-sol transitions in response to AdoB 12 and light, respectively. The chemo-and photo-induced phase transitions enabled encapsulation and controlled release of protein molecules such as the biofilm-degrading glycosyl hydrolase PslG, a potential agent for combatting multidrug-resistant bacterial species in chronic infections. The creation of those uncommon protein architectures promises great opportunities for materials biology and ''smart'' therapeutic delivery.

Genetically engineered materials: Proteins and beyond

“…Since the gel is fully genetically encoded, interesting functions commonly associated with complex biomacromolecules but rare to conventional synthetic materials may be facilely introduced and faithfully preserved throughout the material synthesis. Thanks to the diversity of natural and designed proteins, various stimuliresponsive and functional smart hydrogels have been created using different protein building blocks, such as photoresponsive hydrogels comprising the B 12 -dependent photoreceptor CarH C protein and capable of controlled release of proteins and living cells (Figure 4c), metalloprotein-containing hydrogels for selective sequestration of heavy metals (e.g., oceanic uranium, and chromate), as well as hydrogels containing antimicrobial proteins [86][87][88][89]. One drawback of these genetically engineered hydrogels lies in their weak mechanics, which can be attributed to the large size of the SpyTag/SpyCatcher crosslinker and correspondingly the low crosslinking density.…”

Visible Light‐Responsive Dynamic Biomaterials: Going Deeper and Triggering More

“…Leveraging light‐mediated protein–protein interactions will continue to open new doors in the biomaterials space. [ 104 ] Ruthenium complexes have also been demonstrated in several biomaterial applications, and may continue to be useful in future in vivo experiments as near‐IR‐sensitive crosslinkers. Toxicity concerns appear to be modulatory depending on compound design, though further research is warranted to determine the specific origins of their toxicity and the photocleavage process of these complexes.…”

B 12 -induced reassembly of split photoreceptor protein enables photoresponsive hydrogels with tunable mechanics

“…In addition to being fully genetically encoded, this protein chemistry, alongside its later optimized versions (e.g., Spy002 and Spy003) (19,20), is also noted for its high efficiency and specificity, reminiscent of the powerful Cu (I)catalyzed azide/alkyne cycloaddition click reaction (21)(22)(23). These prototypical genetically encoded click chemistries (GECCs) have found numerous applications in creating advanced protein architectures, subunit vaccines, and functional biomaterials (17,21,(24)(25)(26)(27)(28)(29)(30)(31). On the other hand, since these GECC pairs react spontaneously and invariably form static and rigid covalent adducts, we envisioned that an alternative reaction mode, which is inducible and forms a stable yet dynamically tunable product, would be complementary to the existing GECCs and thus open the door to diverse smart materials.…”