Team:SCAU-China/modeling
molecular simulation
In the design of fusion protein, the order of each protein part and whether linker is needed are important problems. Because this will directly affect whether the fusion protein can operate in the expected way. In our first experiment, because there was no connecting peptide, each part could not complete its function well. Please refer to the engineering page for details. Therefore, in our design, linker peptides are considered necessary. The design of linker peptides must make each functional protein meet the following two conditions: first, the polypeptide chains of the proteins should not affect each other, such as folding and winding together, resulting in the loss of each other's activity; Second, the active centers of them should be far away from each other, which will not form steric hindrance and affect the performance of activity. If the linker peptide is too long, the synergy between the two proteins may be weakened. If it is too short, it may affect the activity of the protein. Only a suitable linker peptide can not only connect the two proteins into a macromolecule, but also will not affect the maintenance of their respective functions (Yan Luying et al., 2008). At present, researchers have also designed a large number of linker peptides with different sequences and structures for the construction of recombinant fusion proteins. According to the function of linker peptides, they are mainly divided into three categories: flexible linker peptides, rigid linker peptides and cleavable linker peptides in vivo. When certain interactions are required between connected functional domains, flexible connecting peptides can be used. They are generally composed of small nonpolar amino acids (such as glycine) or polar amino acids (such as serine or threonine) (GGGGS) n (n ≤ 6) was designed as an excellent flexible linker peptide. However, the soft connecting peptide allows the functional proteins at both ends to swim freely. When forming a side-by-side conformation, this "side-by-side" structure makes the whole fusion protein compact and winding, which is easy to form dimer, resulting in the decline of activity. The rigid linked peptide is not easy to bend, which ensures the relative stability of functional protein spacing. The representative is (EAAAK) n (n ≤ 6) helical sequence, which has a secondary structure α-helix. Another type of rigidly linker peptide is the proline rich sequence (XP) n, x, which can be designed as any amino acid, but preferably alanine, lysine, or glutamate. Other linker peptides include cleavable linker peptides in vivo. It is mostly used in biopharmaceutical, but not in the construction of fusion protein (Yu Jian et al., 2016, Li Jianfang et al., 2015). We used (XX Software) to simulate the spatial structure of the fusion protein with rigid or flexible linker.
Figure 1: Molecular simulation results of fusion protein with or without linker
The results from left to right are no linker, flexible linker and rigid linker. Proteins are labeled in a red to purple pattern from N-terminal to C-terminal.
It can be seen that in the absence of linker and existence of flexible linker, the heavy metal binding protein of the fusion protein is entangled with other structures, and the heavy metal binding protein domain can not play a normal function. After adding rigid linker, it can be found that the domains of each part are effectively separated. This is also the scheme we finally adopted and delivered to the wet lab group for verification.
Recovery time
In the practical application environment, Chlamydomonas will be in the water polluted by heavy metals with the device. Due to the limitation of device space and heavy metal stress, there must be a working limitation. If the limitation is exceeded, the working capacity of the device will be greatly reduced. This limitation is also known as the recovery time of the device. We have summed up two criteria for judging the recovery time. First, the algae group in the device enters the decline stage, which is the cell level standard. Second, the autophagy intensity of algae reached the peak, which was the standard at the molecular level. These standards can be realized by some markers in the device, such as dissolved oxygen, algae density, etc. By measuring these markers, we can quantify the working state of algae and evaluate the working limitation.
References
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- 于健, Setrerrahmane, S., and 徐寒梅. (2016). 连接肽在融合蛋白设计中的选择及应用. 药物生物技术 23, 260-263.
- 李剑芳, 王春娟, and 邬敏辰. (2015). 连接肽的设计及在融合蛋白中的应用. 食品与生物技术学报 34, 1121-1127.
