Part:BBa_K4803015

TVMV^Thr-AI_Stable

This biobrick is a Part of TVMV ^Thr-AI [1] that improves the stability of Thr-CS (Thr-Cleavage Site) by mutating the linker before and after Thr-CS.

Usage and Biology

This part is a linker-connected part of TVMV Protease, Thr-Cleavage Site, and Inhibitor of TVMV Protease. In the presence of Threonine Protease, the linker between TVMV Protease and Inhibitor is cleaved and TVMV Protease is activated. By preparing this part in the cell in advance, TVMV Protease can be activated more than the expression level of Threonine Protease, and thus an amplification effect is expected when viewed from the perspective of the entire system.

Figure 1. The Mechanism of Amplification System

In addition, this Part is expected to suppress the activation of TVMV Protease in the absence of Threonine Protease because it has the highest Cleavage Site stability among the sequences we have evaluated in Dry's simulation by changing the linkers before and after the Thr-Cleavage Site in various ways.

Characterization

In UTokyo2023 project, we modified the linkers around the Inhibitor domain and Cleavage site in about 20 different patterns based on the sequences used in the paper, and compared the prediction accuracy with respect to the original sequences and symmetric sites. Local Colab Fold 1.5.2 [2] was used for protein structure prediction, and pLDDT values were used for comparison of prediction accuracy. In addition, the predicted 3D structures were compared to ensure that the molecule made from the mutated sequence would have the same function as before the mutation.

(a)

(b)

Figure 2. (a) Comparison graphs of pLDDT at each base of TVMV ^Thr-AI and mutated TVMV ^Thr-AI sequences. (b) Comparison of 3D structures predicted by Local Colab Fold.

Reference

[1] Stein, V., & Alexandrov, K. (2014). Protease-based synthetic sensing and signal amplification. Proceedings of the National Academy of Sciences, 111(45), 15934–15939. https://doi.org/10.1073/pnas.1405220111

[2] Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). Colabfold: Making protein folding accessible to all. Nature Methods, 19(6), 679–682. https://doi.org/10.1038/s41592-022-01488-1