To solve the problem, we introduce mutations on C terminal of CRY2, charges on which is probably reasonable for its oligomerization. According to theoretical postulation, positive charge on C terminal facilitates oligomerization while negative charge inhibits. Upon knowing that, the arginine on 489 is mutated to glutamate (R489E) and aspartate (R489D), or amino acids from 489 to 498 are completely deleted(Δ489-498), making the mutant C terminal more negative than wild type. Mutants and its principle is shown in Table1. CRY2wt and its mutants are all tagged with mCherry, in order to show the distribution of CRY2 in cells. Meanwhile, charge of N terminal is critical for CRY2-CIB1 interaction. We also change the N terminal charge of CRY2 to weak its interaction with CIB1 and increase light threshold of the system.

In our project, the core objection is to construct a Phase-separation controlled Downstream enzymatic reaction system, so maximizing the impact caused by the different manipulation is necessary to ensure the high efficiency of the system. The smear distribution inhibits the chance of the contact between enzyme and substrate, compared to aggregated distribution in the phase. As a result, the higher possibility of that smear distribution in the bacteria appear, the lower rate will the overall enzymatic reaction be in the macrograph.

As interaction between mCherry-CRY2 and CIB1-GCN(4)-FUS-GFP is the key of phase formation leading to downstream proteins recruitment, excluding any other influence factors that impedes this hetero-oligomerization should be inhibited. Since CRY2 homo-oligomerization decreases the chance for CRY2 to interact with CIB1 presented in the phase, our improvement is valid.

To promote the efficiency of the system, excluding any other influence factors or regulating hetero-oligomerization are both available ways. One of the most instinctive attempts is to regulate the hetero-oligomerization by mutating the interaction site. The CIB1-binding site of CRY2 locates on the N-terminal of the peptide, ordered as MKMDKKTIV…… Obviously, several acid or basic residues consecutively presents, which contribute to the interaction between CIB1 an CRY2. Further mutation can be exerted to these sites.

As widely used parts in light-controlled system, CRY2 and CIB1 are in the need of improvement. Our improvement eliminates an impede against the smooth manipulation to light-control systems, thus is potentially valuable in various scientific researches and bioengineering. For instance, it can be used to research on pathways, protein functions, or even testing new Phase Separation Components.

Duan, L., et al., Understanding CRY2 interactions for optical control of intracellular signaling. Nat Commun, 2017. 8(1): p. 547.