Red-Shifting B12-Dependent Photoreceptor Protein via Optical Coupling for Inducible Living Materials.

Cobalamin (B12)-dependent photoreceptors are gaining traction in materials synthetic biology, especially for optically controlling cell-to-cell adhesion in living materials. However, these proteins are mostly responsive to green light, limiting their deep-tissue applications. Here, we present a general strategy for shifting photoresponse of B12-dependent photoreceptor CarHC from green to red/far-red light via optical coupling. Using thiol-maleimide click chemistry, we labeled cysteine-containing CarHC mutants with SulfoCyanine5 (Cy5), a red light-capturing fluorophore. The resulting photoreceptors not only retained the ability to tetramerize in the presence of adenosylcobalamin (AdoB12), but also gained sensitivity to red light; labeled tetramers disassembled on red light exposure. Using genetically encoded click chemistry, we assembled the red-shifted proteins into hydrogels that degraded rapidly in response to red light. Furthermore, Saccharomyces cerevisiae cells were genetically engineered to display CarHC variants, which, alongside in situ Cy5 labeling, led to living materials that could assemble and disassemble in response to AdoB12 and red light, respectively. These results illustrate the CarHC spectrally tuned by optical coupling as a versatile motif for dynamically controlling cell-to-cell interactions within engineered living materials. Given their prevalence and ecological diversity in nature, this spectral tuning method will expand the use of B12-dependent photoreceptors in optogenetics and living materials.

B12-induced reassembly of split photoreceptor protein enables photoresponsive hydrogels with tunable mechanics.

Although the tools based on split proteins have found broad applications, ranging from controlled biological signaling to advanced molecular architectures, many of them suffer from drawbacks such as background reassembly, low thermodynamic stability, and static structural features. Here, we present a chemically inducible protein assembly method enabled by the dissection of the carboxyl-terminal domain of a B12-dependent photoreceptor, CarHC. The resulting segments reassemble efficiently upon addition of cobalamin (AdoB12, MeB12, or CNB12). Photolysis of the cofactors such as AdoB12 and MeB12 further leads to stable protein adducts harboring a bis-His-ligated B12. Split CarHC enables the creation of a series of protein hydrogels, of which the mechanics can be either photostrengthened or photoweakened, depending on the type of B12. These materials are also well suited for three dimensional cell culturing. Together, this new protein chemistry, featuring negligible background autoassembly, stable conjugation, and phototunability, has opened up opportunities for designing smart materials.