热稳定性
分子动力学
化学
氢键
突变体
生物物理学
突变
活动站点
螺旋(腹足类)
α螺旋
蛋白质结构
酶
定点突变
蛋白质工程
生物化学
结晶学
计算化学
生物
分子
有机化学
生态学
基因
蜗牛
作者
Thana Sutthibutpong,Triwit Rattanarojpong,Pongsak Khunrae
标识
DOI:10.1080/07391102.2017.1404934
摘要
Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.
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