Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein

Abstract

Membraneless organelles (MLOs) formed via protein phase separation have great implications for both physiological and pathological processes. However, the inability to precisely control the bioactivities of MLOs has hindered our understanding of their roles in biology, not to mention their translational applications. Here, by combining intrinsically disordered domains such as RGG and mussel-foot proteins, we create an in cellulo protein phase separation system, of which various biological activities can be introduced via metal-mediated protein immobilization and further controlled by the water-soluble chlorophyll protein (WSCP)—a remarkably stable, red-light-responsive singlet oxygen generator. The WSCP-laden protein condensates undergo a liquid-to-solid phase transition on light exposure, due to oxidative crosslinking, providing a means to control catalysis within synthetic MLOs. Moreover, these photoresponsive condensates, which retain the light-induced phase-transition behavior in living cells, exhibit marked membrane localization, reminiscent of the semi-membrane-bound compartments like postsynaptic densities in nervous systems. Together, this engineered system provides an approach toward controllable synthetic MLOs and, alongside its light-induced phase transition, may well serve to emulate and explore the aging process at the subcellular or even molecular level.

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production

“…In addition, liquid-tosolid phase transition may restrict the diffusion of substrates into the enzyme-laden protein condensate, thus shutting down the biochemical reactions. [18] A synthetic prion protein domain (sPFD) [19] that is rich in hydrophobic and polar amino acid residues makes the interactions between IDPs proteins more orderly. Thus, to tune the rigidity of the MLOs, sPFD was incorporated into A-IDPs to generate the recombinant sPFD-A-IDPs, and subsequently affect the MLO rigidity.…”

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production

“…MLO could exhibit not only liquid‐like behavior, but also gel‐or amyloid/fiber‐like characteristics. In addition, liquid‐to‐solid phase transition may restrict the diffusion of substrates into the enzyme‐laden protein condensate, thus shutting down the biochemical reactions [18] . A synthetic prion protein domain (sPFD) [19] that is rich in hydrophobic and polar amino acid residues makes the interactions between IDPs proteins more orderly.…”

Photo‐Responsive Phase‐Separating Fluorescent Molecules for Intracellular Protein Delivery

“…Its intracellular disintegration depends on the level of glutathione; the response rate is slow, taking hours to release the payload. Here we aim to develop phase‐separating small molecules to convoy proteins into the cytosol and use light to regulate cargo distribution inside cells [48–53] . A photo‐responsive Norrish carbonyl group was included between the hydrophobic py moiety and the hydrophilic linker (Figure 1a) to allow for photocleavage via the Norrish type II reaction [54–56] .…”