Abstract
Compared with the wild type of lipase (WTL), mutant lipase 6B has twelve mutations (A15S, F17S, A20E, N89Y, G111D, L114P, A132D, M134E, M137P, I157M, S163P, N166Y). The melting temperature of 6B (78.2°C) is much higher than that of WTL (56°C). Hydrogen bond (HB) play an important role in stabilizing the protein. It is important to analyze how mutations affect hydrogen bond and hydrogen bond network and explain how hydrogen bond and hydrogen bond network affect lipase thermostability by the change of the intensity of HB and HB networks with temperature changing. Study the dynamics of HB and HB networks to find that how HBs and HB networks change over time and over temperature in WTL and 6B. Long time MD simulations of WTL and 6B are carried out to analyze how mutations affect hydrogen bond and hydrogen bond network. All proteins were simulated at 300K, 325K, 350K, 375K, 400K for 300ns respectively. The definition of HB is that the distance between acceptor and donor is smaller than a cutoff 3.0 Å and the angle between Donor-H and H-Acceptor is larger than 120o. If two or more HBs connect together, they formed HB network. In the network, residues that formed HB represent nodes, the HB interactions between residues represent edges. The persistence value of HB is computed by . The persistence values of HBs formed by mutations A15S, A20E, G111D, M137P, N166Y are significantly different from that of WTL. HB Glu20-Ser24, Asp111-Asp144, Leu160-Tyr166 and Lys170-Tyr166 are important to stabilize 6B. In addition, the HB networks dynamics show that there are three HB networks are more stable in mutants than that in WTL. The first HB network makes β3, β5, loop and 310-helix closely connect with each other at mutants. The second HB network increases the rigidity of the loop, αC, β3 and β5. The third HB network enhances the interaction between loops, αB and αC. The higher HB persistence value generally means that the HB is more stable. These mutations directly improve the stability of these HBs referring to their persistence values, which show that mutations strengthen the ability of HBs to withstand high temperature and then stabilize the secondary structure. It is thus clear that the mutations change the stability of HBs and the HB networks, which are responsible for increasing protein thermostability.
Published Version
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