Model
Abstract

In order to obtain mutants with better properties, we built protein models and calculated their mutable sites. By scoring the mutated models, we determined which ones were the best mutants. By calculating the energy of the molecules at docking, we can also verify whether the mutants are more active when reacting with the substrate.

Table 1: Mutable structural residues
# Site Original residues Mutational residues
1 18 Lys Arg or Gln
2 20 Gln Arg
3 51 Gln Ala
4 159 Val Phe or Leu
Table 2: Mutable nonstructural resideus
# Site Original residues Mutational residues
1 16 Ser Gly or Asp
2 161 Thr Phe or Leu
3 172 Tyr Ile
Exploration of upper relationship between residues of stem bromelain

The definition of upper relationship means that some single mutations of A are unfavorable, but the addition of B mutation will have better performance than single B mutation, which means that B has upper relationship with A. After calculating by Relax, we found that the Gly single mutation at site 16 had an upper relationship with the Leu single mutation at site 67: when Leu single mutation was detected, the structure was disordered, RM value was 1.4, and energy was -348. But with the addition of Gly, the structure stabilizes and the energy drops to -614.82. At the same time, the single mutation of Gly at site 16 has an upper relationship with the single mutation of Val at site 64: the energy of single mutation of Val is -599, while the energy of mutation of both Gly and Val decreases to -608.

Substrate docking of stem bromelain and its variant
Figure 3: Model of wild-type binding to BAEE
Figure 3 b: Energy of binding(Wild-type)
Figure 3 c: Model of BAEE binding to mutant
Figure 3 d: Energy of binding(Mutant)

After literature review and network search, BAEE was finally determined as the reaction substrate of bromelain, and its docking and energy calculation were performed with bromelain and mutated variants. The calculations were done online by PDBePISA.

Finally, the binding energy of bromelain in wild-type was -125.09, while that in mutant was -126.26, which decreased. This also proves that the stem bromelain variant we developed is more easily bound to the substrate and its reactivity is enhanced.

(Figure 3a, b, c and d)

Molecular Dynamics Simulation Analysis

A molecular dynamics simulation was conducted to analyze the binding stability of Stem Bromelain(212aa) and BAEE complexes, where multiple descriptors were analyzed to understand the flexible and stable nature of the complexes. The system has been balanced in advance (the result of temperature and pressure balance is shown in below.(Figure4 a, b, c and d)

Figure 4 a: the result of temperature balance
Figure 4 b: the result of pressure balance

After the whole system was balanced, the molecular simulation started to run, which took 10h (1ns) in total.

RMSD and RMSF analysis were performed on the simulated results, and the results were shown in the figure below. It can be seen that Stem bromelain(212) has good reactivity with BAEE in neutral environment. The RMSD value was less than 0.4, and the RMSD value structure did not change much before and after simulation, indicating that the reaction between the complex was very stable. At the same time, the RMSF value changed greatly, which reflected that the atomic motion of Stem bromelain (212) was relatively free when it reacted with BAEE.

Figure 4 c: the RMSD of Stem Bromelain(212) and BAEE complex
Figure 4d: the comparing RMSD of Stem Bromelain(212) and BAEE complex