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我们采用并行计算的分子动力学研究了蛋白质的热稳定性,利用蛋白质敲除的技术研究了局部的结构对蛋白质整体结构的热稳定性的影响,另外还通过全电子结构计算来研究蛋白质分子的前线轨道和蛋白质活性和热稳定性的联系.我们的研究工作主要包括三个方面的内容:1.用并行计算分子动力学模拟耐热性邻苯二酚双加氧酶(TC23O)的热稳定性.蛋白质的动力学转变温度的差异,说明了TC23O与同源蛋白1MPY相比有较高的热稳定性.通过分析与中子散射谱相关的指数β和定压热容量Cp,揭示了蛋白质中的228位的脯氨酸残基、盐桥Lys188N-Glu291OE1、金属离子和水溶剂对蛋白质热稳定性的影响存在一定的差异,其中突变体Pro228Ser和Glu291Gly的动力学相变温度分别降低了10℃和19℃,四种金属离子的替换后发现没有显著地影响蛋白质的热稳定性,而水溶剂通过在一定程度上影响蛋白质可能构象的改变过程来改变蛋白质的热稳定性.而所有的结果和已知实验得到的结果有很好的吻合.2.用敲除蛋白质分子的部分片断来研究相应突变体的热稳定性.我们选择的敲除主要有尾部的敲除、无规则卷曲的敲除和α-helix的去除,其中的突变体mut1在尾部切除了从300-319的9个残基,突变体mut2从61-66的无规则卷曲上切除了6个残基,突变体mut3从21-26的包含有部分α-helix的片段上切除6个残基.3.用蛋白质的全电子结构计算研究了前线轨道与蛋白质TC23O和突变体P228S的生物活性以及稳定性的联系.分析前线轨道发现,在野生型突变体P228S中出现的活性位点中心的残基及其能级为TRP_196-10.63526和-9.98105,而突变体P228S中TYR_262-10.66049和TRP_196-9.98779.可以看出在蛋白质的活性中心位点,尽管二者有差距,但能级差距很小.也可能正因为如此,二者在常温下并没有特别明显的酶催化活性的差别.另外在前线轨道里面出现了大量的参与维持蛋白质稳定的盐桥的氨基酸残基,在野生型和突变体P228的前线轨道中出现比例为1/6,说明前线轨道的成分,除了酶活性中心位点的一些残基以外,还有大量的形成盐桥的残基.关键词:并行计算分子动力学,蛋白质敲除,酶活性中心,盐桥,定压热容量,动力学相变温度,全电子结构计算,前线轨道AbstractThe thermostability of protein thermostable cathechol 2,3-dixoygenase (TC23O)and mutants has been studied by the parallel molecular dynamics simulations, farther the different fragments are knocked out to learn how the fragment influence the thermostability of TC23O, finally the linkage of active center and thermostability of protein TC23O and mutant P228S with the frontier orbital energy levels using the method of full electronic calculation.1.The thermostability of protein thermostable cathechol 2,3-dixoygenase (TC23O)has been studied by the parallel molecular dynamics simulations. By analysis ofthe exponent β which is related of scattering spectrum and constant-pressure heatcapacity Cp, we reveal the respective contribution of a specific residue 228proline, a specific salt bridge Arg186NH1-Glu291OE2, four ions and differentwater environment to the thermostability of TC23O. The dynamic transition temperature of the mutants Pro228Ser, Glu291Gly of the TC23O was decreased about 10℃ and 19℃ respectively. The displacement of the four ions we tried have no significant change of the thermostability of TC23O. Water affects the thermostability by influencing the changes of accessible conformation in certain extent. All these results agree with the known experimental results.2.The thermostability of tentative mutants of protein thermostable cathechol2,3-dixoygenase (TC23O) which generated by protein knockout has been studied by the parallel molecular dynamics simulations. The targets of the knockout include C-end fragment, random coil fragment of 61-66 and part of a α -helix fragment of 21-26. By analysis the amplitude of the root mean squared (rms)amplitude of motions〈△U2〉 change with temperature, we find that below the room temperature all of the tentative fragment do not significantly influence the thermostability of protein thermostable cathechol 2,3-dixoygenase (TC23O),C-end fragment does not ,as expected, remarkably influence the thermostability of TC23O with the increasing temperature, which is contrary to the fragment of61-66, the fragment 21-26 only significantly effect the thermostability of TC23O in the temperature range of 330-430K.3.We investigate the linkage of active center and thermostability of protein TC23Oand mutant P228S with the frontier orbital energy levels using the method of fullelectronic calculation. The result indicated that the frontier orbital energy levelsare sensitive to the change of the primary sequence and advanced conformationof protein. We could find some residues of the active center present in the frontierorbits, and about 1/6 molecular orbits are composed of residues which areelements of salt bridge.Key words: parallel molecular dynamics, protein knockout, active center of enzyme,salt bridge, constant-pressure heat capacity, dynamics transition temperature,full electronic calculation, frontier orbit