【作者】 夏元铃；

【导师】 符云新； 柳树群；

【作者基本信息】 云南大学 ， 生物信息学， 2018， 博士

【摘要】 该论文主要进行了两方面研究,分别为:1.静电在枯草杆菌蛋白酶类丝氨酸蛋白酶(subtiIisin-Iike serine protease)温度适应中的作用静电在酶的温度适应中扮演着很重要的角色。为了研究静电在酶温度适应中的可能作用,我们以来源于嗜冷菌Vibriosp.PA-44(VPR)、常温菌Engyodontium album(Tritirachium album)(PRK)和嗜热菌Thermus aquaticus YT-1(AQN)的枯草杆菌蛋白酶类丝氨酸蛋白酶为研究对象,采用多副本分子动力学模拟和连续静电计算方法对三者的静电相互作用强度(包括盐桥、盐桥网络以及钙离子)以及静电表面电势进行了比较性研究。在三个蛋白酶的晶体结构中,AQN所包含的盐桥数量最少,但在分子动力学模拟过程中,AQN中形成了比VPR中更多的新生盐桥,使得在AQN中参与形成盐桥/盐桥网络的带电残基比率高于VPR中的比率。在各自来源物种的生活.温度条件下,酶中所有盐桥的平均静电强度在AQN、PRK和VPR中分别为最强、适中和最弱,表明盐桥总体上对三个蛋白酶热稳定性的贡献分别为最大、适中和最小。实际上,AQN中的绝大多数盐桥均具有较高的负静电自由能值而有助于在高温条件下维持蛋白质的热稳定性,而VPR中约一半的盐桥可以在稳定(负静电自由能值)和去稳定(正静电自由能值)之间相互转换,从而有助于在低温条件下增强其构象柔性。由于单个盐桥、盐桥网络以及钙离子在空间分布以及静电强度上存在差异,它们对这三个蛋白酶的局部结构稳定性/构象柔性会做出不同的贡献。三个蛋白酶的活性中心均被强电负表面电势所覆盖,这可能会保证活性中心的构象柔性,从而有利于催化反应过程中的质子转移和亲核攻击。此外,负电、正电和电中性电势在三个蛋白酶表面,尤其是后表面上,表现出不同的分布,这可能会影响蛋白质的稳定性,同时还有还可能调节其局部构象柔性和刚性。这些结果表明,静电通过调节蛋白质全局或局部结构稳定性以及构象柔性或刚性,可同时对枯草杆菌蛋白酶类丝氨酸蛋白酶的热和冷适应做出贡献。该方面的研究为进一步定点突变和酶工程改造研究奠定了理论基础。2.甲型H3N2流感病毒抗原变化预测方法研究使用甲型流感H3N2的HA1(hemagglutinin 1)蛋白氨基酸序列比较预测其抗原变异可以提高监测流感毒株免疫效应的能力,从而提高疫苗筛选的效率。HA1突变率高且突变的积累会导致病毒抗原持续不断地漂移,给新毒株抗原特征的预测带来很大的挑战。尽管以前的研究在使用氨基酸序列预测抗原变异方面已经取得了很大的进展,但仍有进一步改进的空间。这里,我们提出了一种改进的方法来进一步提高预测甲型H3N2流感抗原变异的准确性,并平衡预测的敏感性和特异性。新方法首先通过两步法鉴定与抗原变异相关的候选关键位点:i)根据其对抗原变异的重要性对HA1区域的329个氨基酸位点进行排序;和ii)在排序好的位点上通过线性多元回归分析获得多组关键位点。随后,在训练集中利用获得的关键位点推测氨基酸距离和位点重要性系数。接着,利用得到的关键位点、关键位点重要性系数以及氨基酸距离构建线性预测模型。最后,使用贝叶斯信息准则(Bayesian information criterion,BIC)选择出最优的预测模型用于抗原特征预测。针对训练集,我们的方法鉴定出11个抗原关键位点,明显少于之前方法鉴定的18-39个位点。将此方法用于包含31 878对抗原对的验证集时,改进的方法在总体上得到了最好的预测效果。更多还原

【Abstract】 This thesis was divivded into two main parts:1.Insights into the role of electrostatics in temperature adaptation of subtilisin-like serine proteasesElectrostatics plays an important role in the temperature adaptability of enzymes.To investigate the possible role of electrostatics in different temperature adaptations,in this section we selected the subtilisin-like serine proteases from psychrophilic Vibrio sp.PA-44(VPR),mesophilic Engyodontium album(Tritirachium album)(PRK),and thermophilic Thermus aquaticus YT-1(AQN)as research objects and performed a comparative study on the electrostatic interaction strengths(i.e.,those of salt bridges,salt-bridge networks,and calcium ions)and electrostatic surface potentials using methods of multiple-replica molecular dynamics(MD)simulations and continuum electrostatics calculations.Although the thermophilic AQN contains the least number of salt bridges among the crystal structures of these three proteases,more newly-formed salt bridges were formed in AQN MD simulations than in VPR simulations,and this results in a higher proportion of charged residues that form salt bridges/salt-bridge networks in AQN than in VPR.The electrostatic strength averaged over all salt bridges in AQN,PRK,and VPR is strongest,moderate,and weakest,respectively,at respective organism growth temperatures,indicating that salt bridges as a whole make the largest,moderate,and least contribution to the thermostability of VPR,PRK,and VPR,respectively.Actually,most salt bridges in AQN exhibit more negative electrostatic free energy values and hence aid in maintaining the protein thermostability at high temperatures,while nearly half of the salt bridges in VPR can interconvert between being stabilizing(negative electrostatic free energy values)and being destabilizing(positive electrostatic free energy values)and hence likely aid in enhancing the conformational flexibility at low temperatures.The individual salt bridges,salt-bridge networks,and calcium ions contribute differentially to local structural stability/conformational flexibility of these three proteases,depending on their spatial distributions and electrostatic strengths.The shared negatively charged surface potential around the active center of the three enzymes may ensure the active-center flexibility and hence benefit to nucleophilic attack and proton transfer.In addition,the differences in distributions of the electro-negative,electro-positive,and electro-neutral potentials,especially over the back surfaces of the three proteases,may affect not only the protein stability but also modulate the local conformational flexibility/rigidity.Our results indicate that electrostatics plays roles in both the heat and coldadaptations of subtilisin-like serine proteases through fine-tuning,either globally or locally,the structural stability and conformational flexibility/rigidity,thus laying the foundation for further mutagenesis and engineering studies.2.A method for predicting antigenic variation in influenza A H3N2Predicting antigenic variation in influenza A H3N2 based on the amino acid sequence comparison will be helpful in improving the capability of evaluating the immune efficacy of influenza vaccines and hence the efficiency of vaccine screening.With respect to the hemagglutinin 1(HA1)of flu virus A,its high mutation rate and mutation accumulation can result in continual anigenic drifts and jumps,making it challenging to predict the antigenic property of a novel strain based on amino acid sequences.Although considerable progress has been made in modeling antigenic variation,there is still a room for improvement in the accurate prediction.In this section,we establish an improved method which can enhance the prediction accuracy of antigenic variation in influenza A H3N2 and provide a better balance between the prediction sensitivity and specificity.In this novel method,first,the key candidate positions associated with the antigenic variation was identified by a two-step process:i)ranking the 329 amino acid sites of HA1 region according to their importance in antigenic variation and,ii)obtaining sets of key positions through multiple linear regression analysis of the ranked positions.Subsequently,the obtained key positions were used to infer optimal amino acid distances and the importance coefficients of key positions,which were used for constructing the final linear prediction model.Finally,the optimal prediction model for antigen feature prediction was selected according to the Bayesian information criterion(BIC).11 amino acid positions optimal for modeling the antigenic variation,which were fewer than the number of key positions(18-39)suggested by previous studies.Most importantly,our method exhibits highest prediction accuracy among the existing methods when applied to the validating data set consisting of 31 878 HI(haemagglutinin inhibition)assays.更多还原