Protocol. 

Membraneless organelles (MLOs) formed via protein phase separation have garnered significant attention recently due to their relevance to cellular physiology and pathology. However, there is a lack of tools available to study their behavior and control their bioactivity in complex biological systems. This chapter describes a new optogenetic tool based on water-soluble chlorophyll protein (WSCP), a red light-induced singlet oxygen-generating protein, to control synthetic MLOs. Upon exposure to red light, WSCP generates singlet oxygen, which triggers the crosslinking of the proteins in the MLOs, resulting in their liquid-to-solid phase transition. The effective delivery of chlorophylls enables the successful reconstitution of WSCP in living cells, thus offering a potential approach to biological regulation at the subcellular level.

Spider silk is recognized for its exceptional mechanical properties and biocompatibility, making it a versatile platform for developing functional materials. In this study, a modular functionalization strategy for recombinant spider silk is presented using SpyTag/SpyCatcher chemistry, a prototype of genetically encoded click chemistry. The approach involves AlphaFold2-aided design of SpyTagged spider silk coupled with bacterial expression and biomimetic spinning, enabling the decoration of silk with various SpyCatcher-fusion motifs, such as fluorescent proteins, enzymes, and cell-binding ligands. The silk threads can be coated with a silica layer using silicatein, an enzyme for silicification, resulting in a hybrid inorganic–organic 1D material. The threads installed with RGD or laminin cell-binding ligands lead to enhanced endothelial cell attachment and proliferation. These findings demonstrate a straightforward yet powerful approach to 1D protein materials.