Secondary structure analysis
5.4. Conclusions
In this chapter we report a new in silico approach for enhancement of thermostability of lipases which can prove to be a guided path for their in vitro evolution, of thermostable proteins efficiently and conveniently. Herein, we discuss improver of thermal stability based on enthalpic contribution, in mesostable triacylglycerol lipase Bacillus subtilis.
Bacillus subtilis lipase A was chosen as the model enzyme to validate the developed tool RankProt as it is an industrially important enzyme. 60 single point mutations were predicted by servers like Cupsat, I-mutant, I-prestab and ERIS to be stabilizing. Out of these 60 mutations combination of 18 combinations of double mutations were ranked higher by RankProt than the wild type structure. Finally, 2 double mutationswere predicted by RankProt to be the most thermostabilizing. The mutations were T47S, Q121N (mut 1) and T47N, Q121N (mut 2). The ranks obtained were 0.5 and 0.54 for mut 1 and mut 2 respectively. Therefore it was predicted that mut 2 will be more stable than mut 1. In silico mutagenesis was performed and the mutated structures were subjected to homology modeling. Molecular superimposition in PyMol showed low RMSD values of 0.278 (mut 1 and 1i6w) and 0.312 (mut 2 and 1i6w), indicating that the wild type and mutated structures were alike.
Physicochemical characterization by RankProt showed that there was increment in γ- turns, salt bridges, ionic interaction, cation pi interaction (CPI), non polar accessible surface area (NASA), main-chain side-chain (MSH) and side-chain side-chain (SSH) hydrogen bonds in mut 1. Again salt bridge (SB), γ-turns (GT and IGT), charged accessible surface area (CASA), hydrophobic interaction (HI), main-chain main-chain (MMH), main-chain side-chain (MSH) and side-chain side-chain (SSH) hydrogen bonds increased in mut 2. It can be said that combinations of increment of different intra-molecular interactions and secondary structures were predicted to stabilize mut 1 and mut 2.
Furthermore molecular docking with (C8) substrate gave lower binding energy for mutants. The binding pocket was intact. This shows that the mutations did not affect the activity of the lipase. Thus mutation did not disturb the catalytic
properties. Contact map analysis of mut 1, mut 2 and the available thermostable structures of Bacillus subtilis lipase highlighted that number of unique contacts in mutants is much higher than the wild type structure. Such unique contacts were more in the loop region of the 3D-structure of the mutants. Furthermore The HBplot analysis of hydrogen bond network in wild type and mutated structures of Bacillus subtilis lipase uncovered that main-chain main-chain hydrogen bonds increased in 4 mutated structures. Main-chain side-chain and side-chain side chain hydrogen bonds increased in all the mutants. Hydrogen bonds <3Å were much greater for the mutants in comparison to the wild type structures. Intramolecular hydrogen bonding networks increased near the β-strand and α-helices of the mesostable lipases. This can result in better packing due to pinning of helices and stands rendering them rigid to unfolding at elevated temperatures.
Molecular dynamics simulation of the wild type and mutants were performed at 320K, 330K and 350K for 30 ns each. The results uncoverd many interesting factors supporting those mutants were more stable than the wild type. The analysis also predicted mut 2 have enhanced stability than mut 1. At higher temperature of 350K the RMSD of mut 2 was much lower than mut 1 and wild type. This reflects that mut 2 is much stable than mut 1 and WT at higher temperatures. Average RMSF plot showed global reduction in flexibility for mut 2 at 350K while mut 1 shows increase.
Appreciable difference of RMSF between wild type and mutants is observed at the N- and C- terminus. The plots illustrating difference in RMSF value between mutants and wild type showed mutants to have lower flexibility at regions where mutations were performed. This observation shows that the mutations have led to decrease in flexibility of the mutants, w.r.t. the wild type. Furthermore lower average Radius of gyration of mutants at all the three temperatures reflects that the mutants are more compact than the wild type. Moreover average number of intra-protein hydrogen bonds was much higher for mut 2 throughout the simulation at different temperatures.
This reflects that mut 2 to have enhanced stability than mut 1 and the wildtype. Again calculation of percentage regular secondary structures and average number of residues showed that they were higher in mutants compared to WT. The mutations
chosen were present in the turns and the average number of residues in turns was more for mutants than wild type at higher temperatures. This shows stability of turns is important for thermostability.
Existing technologies used to thermostabilize proteins rely on the principles of directed evolution and is random. A guided approach is lacking in the existing techniques. This is due to the interplay of various factors in thermostabilizing proteins. Therefore the aforementioned results and methodology reveals that this novel approach will aid in designing thermostabilizing mutations and the guided methodology provided will lead in its achievement with less time, effort, labour and capital. Thus it can revolutionize the method of engineering thermostable proteins in vitro.