Molecular Simulation And Its Application In Bioengineering

Thermophilic enzymes of extremophiles have many theoretical and application merits in studying enzyme evolution, molecular mechanism of protein thermostability and the highest enzyme reaction temperature. After studying the influence of protein structure and function on extremophile thermostability, people can not only describe the physicochemistry principle hidden in protein fold and stability, but also can design novel thermophilic enzymes that can act in high temperature. One of the purposes of studying the factors that influence protein thermostability is to increase the thermostability of mesophilic enzyme. Cyclodextrin glycosyltransferase (CGTase) from Bacillus macerans is an important industrial enzyme in the production of cyclodextrins. However, the working conditions are extreme, which often restrict the usage of CGTase. Besides to screen microorganism for CGTase that fit the requirement of biotechnology, it is also hoped that protein engineering can tailor CGTase to meet demands of industry. During the process of protein engineering, it is needed to understand the knowledge of sequence, function and structure of CGTase.The thermostability of protein CGTase has been studied by the parallel molecular dynamics simulations. We put forward an improved Quantum-behaved particle swarm optimization(QPSO) algorithm for potential calculation, this ensures that all degrees of freedom are adjusted as much as possible before the protein started to move and allows the protein to adjust to the surrounding solvent before performing MD simulation. The structure-stability relationship of CGTase with focus on possible differences in the thermal unfolding pathway is studied by molecular dynamics simulation. The thermostability of the protein CGTase and mutant has been studied by the electrostatic potential optimization and molecular dynamics simulations. And then, we use the heat capacity as the quantitative measure of stability difference between the wild type CGTase and its mutant.The number of local minimizers of potential function increases exponentially with the size of the problem, which characterizes the principal difficulty in minimizing molecular potential energy functions. Due to shortcoming of optimization algorithm that it is often premature convergence, an improved QPSO algorithm is presented by integrating multistage adaptive mechanism. The operator is exerted on the global best position to improve the search ability of the QPSO algorithm. In the experiments, ten functions and general potential energy function are used to validate the performance. The experimental results show that the improved QPSO algorithm has powerful optimizing ability and higher optimizing precision.In this paper for the first time we have performed molecular dynamics simulation of CGTase at high temperatures. And then, we discussed the structure-stability relationship with focus on possible differences in the thermal unfolding pathway. At the same time, we have especially analyzed molecular interactions and intramolecular contacts, in order to search for specific features and structurally important regions, which could explain the difference in thermal stability of the protein during the unfolding process. Distortion of the enzyme is initiated simultaneously in the N-terminal and domain D, while the thermal unfolding of the outer domains of CGTase is faster than that of the catalytic core domain. The catalytic center is well protected by the (α/β)8 TIM-barrel at simulation temperatures up to 600 K. In addition, the unfolding of the 8β-sheets obey the random ordered mechanism, in which theβ-sheets 8, 1 and 6 unfold more rapidly than the others. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. Compared with its initial conformation, internal energy of CGTase increased, accompanied by its conformation transition from a low energy global state to a higher ruleless coil one. The results offer a theoretical basis for explanation of protein degeneration in high temperature process.The thermostability of protein CGTase and mutant has been studied by the molecular dynamics simulations. Root Mean Square Fluctation (RMSF) calculates the mobility of an atom during the MD trajectory for screen mutation site. Considering the charge-residue more character in the mutant, solving linear poisson equation gives the protein's global electrostatic potential, which throw light on the global electrostatic potential optimization feature of CGTase's thermostability. Structural analysis shows that the higher stabilities of the mutant resulted from a increase in the number of salt bridges. Among these salt bridges, Lys88-Glu91, Asp296-Arg335, Arg336-Asp370, Asp145-Arg148 and Arg336-Asp337 are found to be more important for stability. We use the heat capacity as the quantitative measure of stability difference between the wild type and its mutant, the dynamic transition temperature of the mutant is increased about 25℃. Electrostatic mutation method and the heat capacity quantitative measure might offer a viable strategy in the rational design of novel specific protein.Finally, the main contributions in this work are summarized and further research considerations are put forward also.