用同源建模法探索提高脂肪酶的耐热性

分析常温脂肪酶和耐热蛋白质结构的差异,确定影响脂肪酶耐热性的关键氨基酸为Glu,Lys,Ala,Asp,Thr和Gln;继而重构常温脂肪酶的分子,并利用支持向量机分类器预测其耐热性;再用同源建模法模型化。比较重构前后的分子发现:它们的二级结构和三维结构十分相似,但重构后分子中的盐桥和盐桥网络数却明显增加。     

产甲烷常温菌和嗜热菌代谢网络特征的比较研究

以产甲烷的常温古细菌Methanosarcina aeefivorans(MAC)和嗜热古细菌Methanopyrus kandleri(MKA)的代谢网络为研究对象,分析了网络的拓扑结构与功能之间的关联性。本文利用Networkx计算了代谢网络的度、度的中心性、紧密度中心性以及介数中心性等参数值,通过比较分析发现常温古细菌MAC和嗜热古细菌MKA存在氨基酸代谢以及碳水化合物代谢保守途径,且都具有明显更高的度。结果同时表明子途径00010(糖酵解/糖异生)、00020(柠檬酸循环)、00250(丙氨酸、天冬氨酸和谷氨酸代谢)是常温古细菌MAC与嗜热古细菌MKA2个生物体的重要途径。     

氢键与蛋白质耐热性关系的研究

挑选了NCBI COG数据库中具有全基因组的单细胞微生物,选择其中已知三维结构的蛋白质作为研究对象,通过HB- Plus3．0计算出氢键数目并用Perl编程将氢键分类,研究了不同类型的氢键对蛋白质耐热性的影响。结果表明:随着耐热性增高,古细菌类蛋白质中氢键总含量、主链-主链氢键含量,以及非电荷-非电荷氢键含量有显著降低;细菌类蛋白质带电荷-带电荷氢键含量显著增加,而非电荷-非电荷氢键含量显著降低。它们唯一的共同点都是避免在耐热蛋白质中形成非电荷-非电荷氢键。     

环糊精葡萄糖基转移酶N-端热稳定性研究

环糊精葡萄糖基转移酶(CGTase;EC2.4.1.19)是生产环糊精的重要工业用酶,由于工业生产条件的高温加热,使得CGTase的使用受到很大限制。除了筛选合适菌种外,人们更希望利用蛋白质工程手段改造CGTase的热稳定性。文中采用分子动力学模拟取样研究了CGTase的N-端环状区域中氢键与盐桥对其耐热性的影响作用。结果表明:随着模拟温度的升高,非电荷-非电荷氢键占有率显著降低,带电荷-带电荷氢键占有率并没有太大变化,高温下能够保持稳定的氢键几乎都是和带电氨基酸有关;由盐桥的断裂次序的分析,发现在高温下多数盐桥的占有率并没有显著降低,即盐桥的稳定存在对于抵抗高温是有效的。无论是氢键还是盐桥,它们的相同点都是带电荷-带电荷之间的反应受温度影响不大。这一结果说明在蛋白质CGTase中合适的位置增加带电氨基酸数量,增强电荷间静电相互作用,对于提高CGTase的热稳定性是有效的。     

环糊精葡葡萄糖基转移酶热稳定性的研究

环糊精葡萄糖基转移酶(CGTase;EC 2.4.1.19)是一种重要的环糊精生产工业用酶,该转移酶能进行环化、偶合、歧化、水解4种反应.环糊精广泛的用于食品、医药、化妆品、农业和化学工业等生产领域,为了提高环糊精的产量,需要CGTase具有更高的热稳定性.合理的设计耐热蛋白已经成为研究热点.在本文中,首次使用分子动力学模拟研究CGTase热稳定性,以补充实验上不易获得的原子能级和时间相关的信息.使用同源建模方法构建环糊精葡糖基转移酶及其突变体的三维结构,研究氨基酸的突变对CGTase酶耐热性的影响,用CHARMM能量计算CGTase及其突变体的能量与酶蛋白热稳定性之间的关系.证明:氨基酸残基经过突变,突变型比野生型含带电残基更多.相应的,在蛋白天然结构中突变型CGTase中,盐桥数量增加了10%.这些电荷之间、非极性残基之间的非共价键作用力的增强,提高了突变体的刚性,降低了突变体蛋白质分子的总能量,最后增加突变体蛋白质的耐热性.     

磺酰脲类除草剂特点及其环境归趋

磺酰脲类除草剂是一类具有优良毒理和环境特性的超高效除草剂，它作用于乙酰乳酸合成酶（ALS），抑制支链氨基酸－缬氨酸、亮氨酸、异亮氨酸的生物合成，从而抑制细胞分裂和植物生长。磺酰脲类除草剂可被植物的根、茎和叶强烈吸收，并在植株内迅速传导转移和代谢，快速代谢失活是作物对该类除草剂的选择性基础。环境中的磺酰脲化合物主要通过化学水解和微生物降解及少量的光化学分解而消失。     

基于PCA-SVM混合算法的嗜热菌和常温菌网络特征的研究

通过比较嗜热菌和常温菌代谢网络的特征参数,可以从系统角度确定微生物嗜热性的主要因素。本文首先利用主成分分析法对22个网络特征进行相关性分析,根据特征值、载荷值的大小最终选择了11个主要网络特征;用选出的这11个网络特征组成特征向量,利用支持向量机构建分类器,对嗜热菌和常温菌进行分类,其全局平均预测率为82.93%,对常温菌和嗜热菌的平均预测率分别为87.86%和72.40%。结果表明利用主成分分析法选择的网络特征可以很好的表征嗜热菌和常温菌的耐热性,因此簇大小分布的平均信息等11个网络特征是影响微生物耐热性的关键的代谢网络特征因素。     

基于SVM和KNN的蛋白质耐热性分类

以氨基酸含量为特征向量,研究了SVM和KNN预测蛋白质耐热性的准确度。结果表明,基于SVM的分类效果较好,其局部预测率和全局预测率分别为82.4%和83.4%;而基于KNN方法的局部预测率和全局预测率分别为77.6%和79.9%。两种方法的预测率均表明氨基酸含量是影响蛋白质耐热性的主要因素。     

碘酰脲类除草剂特点及其环境归趋

磺酰脲类除草剂是一类具有优良毒理和环境特性的超高效除草剂，它作用于乙酰乳酸合成酶（ＡＬＳ），抑制支链氨基酸－缬按酸、亮氨酸、异亮氨酸的生物合成，从而抑制细胞分裂和植物生长。磺酰脲类除草剂可被植物的根、茎和叶强烈吸收，并在植株内迅速传导转移和代谢，快速代谢失活是作物对该类除草剂的选择性基础。环境中的磺酰脲化合物主要通过化学水解和微生物降解及少量的光化学分解而消失。     

基于复杂网络理论的产甲烷常温菌和嗜热菌的代谢网络的拓扑与模块化的研究

近几年,复杂网络的研究正成为广泛关注的热点,代谢网络是复杂网络的一个例子。本文以产甲烷的常温古细菌Methanosarcina acetivorans(M.acetivorans)和嗜热古细菌Methanopyrus kandleri(M.kandleri)的代谢网络为对象,从拓扑参数以及模块化两方面进行比较研究。结果表明:M.acetivorans与M.kandleri的代谢网络均具有较高的模块化结构。同时发现它们模块化后的代谢网络中的Hub模块均属于氨基酸代谢和碳水化合物代谢,表明这些网络模块均具有一定的功能意义。最后将Hub模块与最紧密的k-核心网络相比较,发现它们节点完全相同,此结果表明代谢网络的最紧密k-核心网络部分也是不同网络比较的重要因素。     

基于氨基酸组成预测蛋白质热稳定性的v-支持向量机方法

支持向量机有许多优点有效防止过拟和,适合大的特征空间,给定数据集的信息压缩.本文首次利用支持向量机从氨基酸组成来预测蛋白质的稳定性.总预测率可以达到80.64%,对嗜热蛋白质的预测率为82.50%,对嗜温蛋白质的预测率为80.29%从预测率可以验证氨基酸组成与蛋白质热稳定性成正相关的关系,支持向量机可以成为基于氨基酸组成预测蛋白质热稳定性的有效工具.     

基于K-近邻算法预测蛋白质热稳定性的研究

基于一级结构信息预测蛋白质热稳定性,对于利用计算机筛选热稳定性蛋白具有重要意义。本文采用k-近邻算法从序列出发预测蛋白质的热稳定性,用自一致性检验、交叉验证和独立样本测试等三种方法评估。仅用20种氨基酸组成作为特征变量时,识别的正确率分别可达100%,87.7%和89.6%;而引入8个新变量后,其精度分别为100%,89.6%和90.2%,对小蛋白质分子识别的精度提高了2.4%。同时探讨了蛋白质分子大小对识别效果的影响。     

基于支持向量机识别嗜热和常温蛋白的研究

采用支持向量机对嗜热和常温蛋白进行模式识别并和偏最小二乘回归比较。结果表明,当惩罚因子C为1,核函数选取线性函数,不敏感常数epsilon取0．01时,经320组数据训练,支持向量机预测的平均正确率为84．9％,后者为86．8％。经1720组数据训练,支持向量机对嗜热蛋白预测正确率达97．4％,对常温蛋白预测的正确率为84．2％,平均90．8％。建立了一种基于序列的识别嗜热和常温蛋白的新方法。     

Insights into thermal stability of thermophilic nitrile hydratases by molecular dynamics simulation

Thermal stability is of great importance for industrial enzymes. Here we explored the thermal-stable mechanism of thermophilic nitrile hydratases (NHases) utilizing a molecular dynamic simulation. At a nanosecond timescale, profiles of root mean square fluctuation (RMSF) of two thermophilic NHases, 1UGQ and 1V29, under enhancing thermal stress were carried out at 300 K, 320 K, 350 K and 370 K, respectively. Results showed that the region A1 (211-231 aa) and A2 (305-316 aa) in 1UGQ, region B1 (186-192 aa) in 1V29, and most of terminal ends in both enzymes are hyper-sensitive. Salt-bridge analyses revealed that in one hand, salt-bridges contributed to maintaining the rigid structure and stable performance of the thermophilic 1UGQ and 1V29; in the other hand, salt-bridges involved in thermal sensitive regions are relatively weak and prone to be broken at elevated temperature, thereby cannot hold the stable conformation of the spatial neighborhood. In 1V29, region A1 was stabilized by a well-organized hook-hook like cluster with multiple salt-bridge interactions, region A2 was stabilized by two strong salt-bridge interactions of GLU52-ARG332 and GLU334-ARG332. In 1UGQ, the absence of a charged residue decreased its thermal sensitivity of region B1, and the formation of a small beta-sheet containing a stable salt-bridge in C-beta-terminal significantly enhanced its thermal stability. By radius of gyration calculation containing or eliminating the thermal sensitive regions, we quantified the contribution of thermal sensitive regions for thermal sensitivity of 1UGQ and 1V29. Consequently, we presented strategies to improve thermal stability of the industrialized mesophilic NHase by introducing stable salt-bridge interactions into its thermal sensitive regions.     

Contribution of main chain and side chain atoms and their locations to the stability of thermophilic proteins

Proteins belonging to the same class, having similar structures thus performing the same function are known to have different thermal stabilities depending on the source— thermophile or mesophile. The variation in thermo-stability has not been attributed to any unified factor yet and understanding this phenomenon is critically needed in several areas, particularly in protein engineering to design stable variants of the proteins. Toward this motive, the present study focuses on the sequence and structural investigation of a dataset of 373 pairs of proteins; a thermophilic protein and its mesophilic structural analog in each pair, from the perspectives of hydrophobic free energy, hydrogen bonds, physico-chemical properties of amino acids and residue–residue contacts. Our results showed that the hydrophobic free energy due to carbon, charged nitrogen and charged oxygen atoms was stronger in 65% of thermophilic proteins. The number of hydrogen bonds which bridges the buried and exposed regions of proteins was also greater in case of thermophiles. Amino acids of extended shape, volume and molecular weight along with more medium and long range contacts were observed in many of the thermophilic proteins. These results highlight the preference of thermophiles toward the amino acids with larger side chain and charged to make up greater free energy, better packing of residues and increase the overall compactness.     

Comparative structural studies of psychrophilic and mesophilic protein homologues by molecular dynamics simulation

Comparative molecular dynamics simulations of psychrophilic type III antifreeze protein from the North-Atlantic ocean-pout Macrozoarces americanus and its corresponding mesophilic counterpart, the antifreeze-like domain of human sialic acid synthase, have been performed for 10 ns each at five different temperatures. Analyses of trajectories in terms of secondary structure content, solvent accessibility, intramolecular hydrogen bonds and protein–solvent interactions indicate distinct differences in these two proteins. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their growth temperatures. However at higher temperatures psychrophilic protein shows increased overall flexibility than its mesophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are non-overlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar directions of motion of each protein especially at 298 K, 310 K and 373 K. Overall, the psychrophilic protein leads to increased conformational sampling of the phase space than its mesophilic counterpart.Our study may help in elucidating the molecular basis of thermostability of homologous proteins from two organisms living at different temperature conditions. Such an understanding is required for designing efficient proteins with characteristics for a particular application at desired working temperatures.     

Comparative structural studies of psychrophilic and mesophilic protein homologues by molecular dynamics simulation

Comparative molecular dynamics simulations of psychrophilic type III antifreeze protein from the North-Atlantic ocean-pout Macrozoarces americanus and its corresponding mesophilic counterpart, the antifreeze-like domain of human sialic acid synthase, have been performed for 10 ns each at five different temperatures. Analyses of trajectories in terms of secondary structure content, solvent accessibility, intramolecular hydrogen bonds and protein–solvent interactions indicate distinct differences in these two proteins. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their growth temperatures. However at higher temperatures psychrophilic protein shows increased overall flexibility than its mesophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are non-overlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar directions of motion of each protein especially at 298 K, 310 K and 373 K. Overall, the psychrophilic protein leads to increased conformational sampling of the phase space than its mesophilic counterpart.Our study may help in elucidating the molecular basis of thermostability of homologous proteins from two organisms living at different temperature conditions. Such an understanding is required for designing efficient proteins with characteristics for a particular application at desired working temperatures.