大鼠尿激酶SYBR Green I real time RT-PCR引物的设计

设计用于SYBR Green I法实时定量逆转录多聚酶链反应(QRT-PCR)检测大鼠尿激酶型纤溶酶原激活因子(uPA)mRNA的引物。从基因库获取靶基因及相关序列,充分收集争分析相关生物信息学数据,应用Oligo 6.22设计出一对长度为21bp的引物,其GG含量为52.4%;上下游引物3’最稳定二聚体和及发夹结构的能量分别为-1.5、-0.40 kcal/mol和-3.5、-O.90 kcal/mol,引物间最稳定二聚体为-3.1 kcal/mol。5’端和中间△G值较高,高于3’端△G;引发效率分别455和403。实验证明,该引物能够高效、特异地实现对靶序列的检测,适用于SYBR Green I法实时定量检测(uPA)mRNA。     

基于新杂合进化算法的蛋白质折叠计算

蛋白质能量最小化是蛋白质折叠的重要内容。用于蛋白质折叠的新的杂合进化算法结合了交叉和柯西变异。基于toy模型的蛋白质能量最小化算例表明,这个新的杂合进化算法是有效的。     

次氯酸与不饱和脂肪酸氧化机制和转化产物的理论研究

摘要 目的：揭示次氯酸与不饱和脂肪酸的氧化反应机制及转化产物。方法：运用Gaussian 16软件包,采用密度泛函方法M06-2X(D3),结合6-31+G(d)基组,在SMD液相水模型水平下进行计算。结果：次氯酸与单不饱和脂肪酸油酸的氧化反应是先形成氯鎓离子中间体,氯鎓离子再与水分子反应生成氯醇,第一步氯鎓离子的形成是控速步骤,其反应活化自由能~8 kcal/mol。环氧化合物和短链的醛是两种转化产物,前者由氯醇脱氯化氢而来,而后者由环氧化合物和氯醇通过系列与次氯酸根的反应而得到,生成它们的控速步骤的反应活化自由能分别为23 和24 kcal/mol。选取两个乙基为取代基的乙烯为油酸模型,其与次氯酸反应的活化自由能仅比油酸高1 kcal/mol。计算得到次氯酸与亚油酸、顺-9,反-11 亚油酸、梓树酸和花生四烯酸模型氧化反应生成氯醇的活化自由能分别是~10、13、16和14 kcal/mol。结论：氯鎓离子中间体机制是次氯酸与不饱和脂肪酸氧化反应的主要机制,反应的活化自由能通常低于15 kcal/mol,意味着此氧化反应动力学上容易发生。氧化产物氯醇能转化为环氧化合物和短链的醛,但活化自由能较高,约23和24 kcal/mol。选取距离双键3个碳以内的结构为不饱和脂肪酸模型,它能够很好地反映不饱和脂肪酸的反应活性。     

丁香油酚和乙酸香茅酯对蜂王信息素活性的影响:量子化学研究

植物次生代谢物质可影响昆虫信息素的功能,但有关影响机制尚不清楚。本文利用量子化学理论分析了广泛存在于花粉或花蜜中的挥发性丁香油酚和乙酸香茅酯能否与蜜蜂蜂王信息素挥发性成分4-羟基-3-甲氧苯乙醇(HVA)反应。使用Gaussian 09软件来完成几何优化、过渡态搜索、频率分析,并计算了反应能垒和速率常数。结果表明,丁香油酚和HVA分别与OH自由基反应生成有机自由基后,通过自由基-自由基途径发生聚合反应(反应能垒为0.613077 kcal/mol,反应速率常数为9.559953×1011cm3/molecule/s),而不易通过自由基-分子途径发生反应(反应能垒为31.792769 kcal/mol,反应速率常数为4.268854×10-13cm3/molecule/s)。相似地,乙酸香茅酯和HVA分别与OH自由基反应后,也可通过自由基-自由基途径发生反应(反应能垒为2.086469 kcal/mol,反应速率常数为2.328216×1011cm3/molecule/s),但不易通过自由基-分子途径发生反应(反应能垒为25.881002 kcal/mol,反应速率常数为1.513828×10-8cm3/molecule/s)。由于全球环境变化可能导致大气中OH自由基浓度升高,使得花蜜或花粉中挥发性不饱和化合物有可能影响蜂王信息素的功能,从而干扰蜂群的化学通讯。     

大鼠尿激酶SYBR Green Ⅰ real time RT—PCR引物的设计

设计用于SYBR Green Ⅰ法实时定量逆转录多聚酶链反应（QRT—PCR）检测大鼠尿激酶型纤溶酶原激活因子（uPA）mRNA的引物。从基因库获取靶基因及相关序列,充分收集和分析相关生物信息学数据,应用Oligo 6．22设计出一对长度为21bp的引物,其GC含量为52．4％;上下游引物3’最稳定二聚体和及发夹结构的能量分别为-1．5、-0．40kcal／mol和-3．5、-0．90kcal／mol,引物间最稳定二聚体为-3．1kcal／mol。5’端和中间AG值较高,高于3’端AG;引发效率分别455和403。实验证明,该引物能够高效、特异地实现对靶序列的检测,适用于SYBR GreenⅠ法实时定量检测（uPA）mRNA。     

1991-2015年我国十五省（区）农民膳食能量及宏量营养素摄入的变化趋势及其人口学特征

目的：分析中国15省（区）农民膳食能量和宏量营养素摄入现状和变化趋势。方法：利用“中国健康与营养调查” 1991—2015年间膳食随访数据,以18岁及以上的农民作为研究对象。采用连续3d 24h膳食回顾法和家庭称重记账法收集膳食资料,借助食物成分表将食物消费量转换成能量及各类营养素摄入量。结果：2015年我国15省（区）农民能量摄入量为1 891.5kcal/d,蛋白质、脂肪和碳水化合物的平均摄入量分别为55.4、66.0、245.2g/d,其供能比分别为12.0%、33.2%和54.0%。与1991年相比,能量、蛋白质、碳水化合物摄入量分别下降了521.7kcal/d、13.7g/d、152.0g/d,脂肪摄入量增加了18.1g/d,蛋白质供能比基本持平,碳水化合物供能比减少了10.8%,脂肪供能比增加了10.6%。2015年,65岁及以上农民的能量、蛋白质和脂肪摄入量均低于其他年龄组,碳水化合物的摄入量随年龄组的升高而降低,低收入农民的能量和碳水化合物摄入量高于中、高收入农民。谷类食物、其他食物、食用油分别为能量、蛋白质、脂肪的主要来源。结论：中国15省（区）农民蛋白质和碳水化合物供能比合理,脂肪供能比较高,应根据农民不同的收入水平和生理条件开展针对性的营养指导。     

基于FoldX力场的蛋白突变建模方法可用于人工锌指酶辅助设计

锌指酶是一种可对动植物基因组内特异序列进行高效遗传修饰的人工重组核酸酶.目前,获取一对有活性的人工锌指酶的常规方法是通过构建大量的锌指蛋白突变体,利用细菌杂交方法从中筛选有结合活性的个体,工作量庞大、专业性强,普通研究人员往往难以胜任,极大地限制了锌指酶技术的广泛应用.因此,采用计算机辅助人工锌指酶设计的方法降低筛选难度和工作量显得十分有必要.本研究选用FoldX分子力场软件对国际锌指酶协会研究人员采用OPEN方法构建的共420个锌指蛋白突变体进行蛋白-DNA复合体建模,并计算模型中锌指蛋白与靶标DNA的结合自由能.经统计分析发现,模拟复合体中的锌指蛋白-DNA结合自由能与该锌指蛋白在哺乳动物细胞内形成无活性锌指酶的概率存在明显相关关系.当结合自由能低于？13.132kcal/mol时形成无活性锌指酶的概率可降低至5%以下,而当结合自由能大于？5kcal/mol时形成无活性锌指酶的概率高达40%.因此,采用FoldX构建锌指蛋白-DNA复合体模型的方法可以辅助缩小锌指蛋白的筛选范围,提高人工锌指酶设计的成功率.     

贵州地方山羊品种H-FABP基因第3外显子SNP研究

[目的]研究3种贵州地方山羊品种H-FABP基因第3外显子的多态性以及进行该基因多态性生物信息学分析,以期筛选出适合的突变位点,研究其与山羊生长性状关联性影响。[方法]以3种贵州地方山羊品种为试验材料构建DNA池,结合直接测序技术筛选H-FABP基因SNPs,并利用在线软件分析H-FABP基因RNA二级结构及其蛋白二、三级结构。[结果]仅在黔北麻羊和贵州白山羊H-FABP基因第3外显子处分别筛选到T120A和G152C两个SNPs,其中exon3-T120A为导致编码氨基酸发生的Phe→Ile改变的错义突变,而exon3-G152C为苏氨酸(Thr)的同义突变。[结论]exon3-T120A位点影响黔北麻羊H-FABP基因RNA二级结构,最小自由能由-189.40 kcal/mol变为-190.00 kcal/mol,其蛋白二级结构无规则卷曲由49变为48,延伸链由36变为37,exon3-G152C位点仅影响贵州白山羊RNA二级结构,最小自由能变为-185.40 kcal/mol。     

海莲、秋茄两种红树群落能量的研究

 本文应用热值测定,对中国两种典型红树群落,即海南岛的海莲群落及福建九龙江口的秋茄群落样品热值、群落能量现存量、能量固定量以及太阳能转化效率进行了测定和分析．结果表明：(1)红树群落各组分样品之间热值有一定的差异;一般叶、花的热值较高,而根、树皮的热值较低。(2)群落能量现存量海莲群落(1984年1月)高达178,627kcal／m2,秋茄群落为70,547kcal／m2,群落能量在不同组分以及不同高度层次上均有不同的分配比。(3)海莲群落(1983)、秋茄群落(1982)能量年净固定量分别为15,772kcal／m2和10,456kcal/m2,相应地对林地太阳光合有效辐射能的转化效率依次为3.01％和2.01％;红树群落比其他植物群落具有较高的能量固定能力及太阳能转化效率。     

脱氮硫杆菌硫化合物载体SoxYZ蛋白的同源建模和结构分析

脱氮硫杆菌(Thiobacillus denitrificans)中的Sox蛋白在硫代谢过程中起着至关重要的作用,硫化合物需先与硫氧化基因族(sox)编码的蛋白质Sox YZ二聚体共价连接后才能与其他酶发生相互作用。利用同源建模法构建硫化合物载体Sox YZ蛋白的二聚体结构并验证了其合理性。二聚体相互作用分析发现Sox YZ蛋白的溶剂可及表面积(solvent accessible surface,SAS)为10 922.92,疏水率为50.85%;亚基Sox Y和Sox Z界面处共含有12个氢键和1个Π键来维持其三维结构的稳定性;二聚体表面呈现明显的正负电势互补,两亚基界面处氨基酸残基的VDW作用能和静电作用能分别为-80.925 13kcal/mol和-323.856 57kcal/mol,这说明静电作用是二聚体形成的主要驱动力;Sox Z亚基的残基Thr28、Arg31、Lys32、Ser64、Gly65、Val66、Ser67对Sox Y亚基活性位点构象的稳定有重要作用。     

Accurate PDZ/Peptide Binding Specificity with Additive and Polarizable Free Energy Simulations

PDZ domains contain 80–100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.     

Influenza viral hemagglutinin complicated shape is advantageous to its binding affinity for sialosaccharide receptor

Do the complexity and the bulkiness of a protein affect the affinity between protein and ligand? We attempted to investigate this problem by using ab initio fragment molecular orbital (FMO) method to calculate the binding energy between human influenza viral hemagglutinin (HA) and human oligo-saccharide receptor. We compared the binding energies of 4 different sizes of human A virus HA H3 subtype complexed with human receptor Neu5Ac(alpha2-6)Gal as a model. The full shape receptor binding domain complexed with Neu5Ac(alpha2-6)Gal had the highest binding energy 170.3kcal/mol at the FMO-HF/STO-3G level, which was 52.3kcal/mol higher than that of the smallest domain-receptor complex. These data provide the consideration of the backyard bulkiness beyond the binding site of protein to the protein-ligand stability.     

Reaction mechanism of caspases: insights from QM/MM Car-Parrinello simulations

Caspases are fundamental targets for pharmaceutical interventions in a variety of diseases involving disregulated apoptosis. Here, we present a quantum mechanics/molecular mechanics Car-Parrinello study of key steps of the enzymatic reaction for a representative member of this family, caspase-3. The hydrolysis of the acyl-enzyme complex is described at the density functional (BLYP) level of theory while the protein frame and solvent are treated using the GROMOS96 force field. These calculations show that the attack of the hydrolytic water molecule implies an activation free energy of ca. DeltaF(A) approximately equal 19 +/- 4 kcal/mol in good agreement with experimental data and leads to a previously unrecognized gem-diol intermediate that can readily (DeltaF(A) approximately equal 5 +/- 3 kcal/mol) evolve to the enzyme products. Our findings assist in elucidating the striking difference in catalytic activity between caspases and other structurally well-characterized cysteine proteases (papains and cathepsins) and may help design novel transition-state analog inhibitors.     

Derivation of class II force fields. VI. Carbohydrate compounds and anomeric effects

The methodology for deriving class II force fields has been applied to acetal, hemiacetal, and carbohydrate compounds. A set of eighteen model compounds containing one or more anomeric centers was selected for generating the quantum mechanical energy surface, from which the force field was derived and the functional form assessed. The quality of the fit was tested by comparing the energy surface predicted by the force field with ab initio results. Structural, energetic, and dynamic properties (vibrational frequencies) were analyzed. In addition, α and β anomeric equilibrium structures and energies of 2-methoxytetrahydropyran, 2-deoxyribose, and glucose were computed at the HF/6-31G* and higher ab initio levels. These calculations provide test data from molecules outside the training set used to derive the force field. The quantum calculations were used to assess the ability of the class II force field and two quadratic diagonal (class I) force fields, CVFF, and Homans' extension of the AMBER force field, to account for the anomeric effects on the structural and energetic properties of carbohydrate systems. These class I force fields are unable to account for observed structural and energetic trends, exhibiting deviations as large as 5 kcal/mol in relative energies. The class II force field, on the other hand, is shown to reproduce anomeric structural as well as energetic differences. An energy component analysis of this force field shows that the anomeric differences are dominated by torsional energies, although coupling terms, especially angle/torsion, also make significant contributions (roughly 1 kcal/mol in glucose). In addition, the force field accurately accounts for both anomeric and exo-anomeric energy differences in 2-methoxytetrahydropyran, and anomeric energy differences in 2-deoxyribose and glucose. © 1998 John Wiley & Sons, Inc. Biopoly 45: 435–468, 1998     

Contributions to the binding free energy of ligands to avidin and streptavidin

The free energy of binding of a ligand to a macromolecule is here formally decomposed into the (effective) energy of interaction, reorganization energy of the ligand and the macromolecule, conformational entropy change of the ligand and the macromolecule, and translational and rotational entropy loss of the ligand. Molecular dynamics simulations with implicit solvation are used to evaluate these contributions in the binding of biotin, biotin analogs, and two peptides to avidin and streptavidin. We find that the largest contribution opposing binding is the protein reorganization energy, which is calculated to be from 10 to 30 kcal/mol for the ligands considered here. The ligand reorganization energy is also significant for flexible ligands. The translational/rotational entropy is 4.5-6 kcal/mol at 1 M standard state and room temperature. The calculated binding free energies are in the correct range, but the large statistical uncertainty in the protein reorganization energy precludes precise predictions. For some complexes, the simulations show multiple binding modes, different from the one observed in the crystal structure. This finding is probably due to deficiencies in the force field but may also reflect considerable ligand flexibility.     

Protein structure prediction based on fragment assembly and parameter optimization

We propose a novel method for ab-initio prediction of protein tertiary structures based on the fragment assembly and global optimization. Fifteen residue long fragment libraries are constructed using the secondary structure prediction method PREDICT, and fragments in these libraries are assembled to generate full-length chains of a query protein. Tertiary structures of 50 to 100 conformations are obtained by minimizing an energy function for proteins, using the conformational space annealing method that enables one to sample diverse low-lying local minima of the energy. Then in order to enhance the performance of the prediction method, we optimize the linear parameters of the energy function, so that the native-like conformations become energetically more favorable than the non-native ones for proteins with known structures. We test the feasibility of the parameter optimization procedure by applying it to the training set consisting of three proteins: the 10-55 residue fragment of staphylococcal protein A (PDB ID 1bdd), a designed protein betanova, and 1fsd.     

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry.

The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, delta delta G0d, produced by the mutations in the pH range 5.0-7.0 varied from 1.9 kcal mol-1 (apparent stabilization) for H124L to -2.8 kcal mol-1 (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between delta delta G0d and the corresponding values for delta delta Hd and T delta delta S0d, the latter quantities being in most cases much larger in magnitude than delta delta G0d. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X-ray crystallography.     

Rotamer strain energy in protein helices - quantification of a major force opposing protein folding

It is widely believed that the dominant force opposing protein folding is the entropic cost of restricting internal rotations. The energetic changes from restricting side-chain torsional motion are more complex than simply a loss of conformational entropy, however. A second force opposing protein folding arises when a side-chain in the folded state is not in its lowest-energy rotamer, giving rotameric strain. chi strain energy results from a dihedral angle being shifted from the most stable conformation of a rotamer when a protein folds. We calculated the energy of a side-chain as a function of its dihedral angles in a poly(Ala) helix. Using these energy profiles, we quantify conformational entropy, rotameric strain energy and chi strain energy for all 17 amino acid residues with side-chains in alpha-helices. We can calculate these terms for any amino acid in a helix interior in a protein, as a function of its side-chain dihedral angles, and have implemented this algorithm on a web page. The mean change in rotameric strain energy on folding is 0.42 kcal mol-1 per residue and the mean chi strain energy is 0.64 kcal mol-1 per residue. Loss of conformational entropy opposes folding by a mean of 1.1 kcal mol-1 per residue, and the mean total force opposing restricting a side-chain into a helix is 2.2 kcal mol-1. Conformational entropy estimates alone therefore greatly underestimate the forces opposing protein folding. The introduction of strain when a protein folds should not be neglected when attempting to quantify the balance of forces affecting protein stability. Consideration of rotameric strain energy may help the use of rotamer libraries in protein design and rationalise the effects of mutations where side-chain conformations change.     

Evaluation of carbohydrate molecular mechanical force fields by quantum mechanical calculations

A quantitative evaluation of 20 second-generation carbohydrate force fields was carried out using ab initio and density functional methods. Geometry-optimized structures (B3LYP/6-31G(d)) and relative energies using augmented correlation consistent basis sets were calculated in gas phase for monosaccharide carbohydrate benchmark systems. Selected results are: (i). The interaction energy of the alpha-d-glucopyranose.H(2)O heterodimer is estimated to be 4.9 kcal/mol, using a composite method including terms at highly correlated (CCSD(T)) level. Most molecular mechanics force fields are in error in this respect; (ii). The (3)E envelope (south) pseudorotational conformer of methyl 5-deoxy-beta-d-xylofuranoside is 0.66 kcal/mol more stable than the (3)E envelope (north) conformer and the alpha-anomer of methyl d-glucopyranoside is 0.82 kcal/mol more stable than the beta-anomer; (iii). The relative energies of the (gg, gt and tg) rotamers of methyl alpha-d-glucopyranoside and methyl alpha-d-galactopyranoside are (0.13, 0.00, 0.15) and (0.64, 0.00, 0.77) kcal/mol, respectively. The results of the quantum mechanical calculations are compared with the results of calculations using the 20 second-generation carbohydrate force fields. No single force field is consistently better than the others for all the test cases. A statistical assessment of the performance of the force fields indicates that CHEAT(95), CFF, certain versions of Amber and of MM3 have the best overall performance, for these gas phase monosaccharide systems.