蛋白质结构型的定义和识别

提出紧结构域的概念 ,由二级结构序列中一段或几段连续的α螺旋和 β折叠构成的空间紧密堆集的最大折叠体称为紧结构域 .利用 3种紧结构域 (α域 ,β域和α/β域 )定义球蛋白的 5种结构型 :α型蛋白 ,β型蛋白 ,α/β型蛋白 ,多域蛋白和 ζ型蛋白 .将 12 6 1个代表性的蛋白质 (10 2 2家族 )进行分类 ,并和SCOP库的分类做了比较 .进行了删去序列冗余的分析 .在此基础上提出结构型的预测方案 ,成功率在 82 %～ 85 %.

蛋白质结构型的识别方法

给出了α型、β型、α/ β型、多域型蛋白质二级结构主序列六联体的分布规律 .提出了根据蛋白质二级结构主序列对蛋白质结构型进行识别 (分类 )的方法 .以蛋白质二级结构主序列三联体为参数 ,利用Mahalanobis距离方法对上述 4种结构型的蛋白质进行识别 ,分类的总体准确率为 81% ;以二级结构主序列中六联体的频数构成蛋白质结构的多样性源 ,利用多样性增量极小化对上述 4种结构型进行识别 ,分类的总体准确率为 83% .

部分小麦低分子量谷蛋白亚基二级结构的预测与分析

为了从蛋白质高级结构的水平上研究小麦低分子量谷蛋白结构与功能的关系,利用互联网上开放的预测软件和蛋白质序列分析软件对已获得全序列,并明确其染色体定位的19个LMW-GS基因的推导氨基酸序列进行二级结构的预测和分析。结果表明,在整个LMW-GS二级结构中,无规则卷曲最多,达69.51%,α-螺旋(28.97%)较β-折叠(1.36%)占有绝对优势,因而归属于α结构型蛋白。其中,信号肽由大量α-螺旋和微量无规则卷曲组成,N-端和重复区均由无规则卷曲占据,而C-末端除富含α-螺旋和无规则卷曲外,还是β-折叠的唯一分布区。同时,两个分子间二硫键存在于无规则卷曲中,而另有6个分子内二硫键分布于α-螺旋内或其附近,由此推导出小麦LMW-GS二级结构的平面模式图。这种特异的LMW-GS二级结构不仅进一步证明了“蛋白质一级结构决定高级结构”,以及信号肽引导新合成的LMW-GS顺利进入相应细胞器等经典理论,而且从空间结构的水平上阐释了LMW-GS多肽链致密化及其互聚体形成、富集,并与HMW-GS一起影响面粉加工品质的结构基础。

Expression,Purification and Activity Detection of VP1 of A-type FMDV

[Objective] The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method] The fragment coding VP1 was amplified by PCR and doubly digested with BamH Ⅰ and XhoⅠ,then cloned into expression vector pGEX-4T-1 and pPROExHTb respectively to get recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1.The recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1 was transformed into E.coli BL21(DE3)and induced by IPTG,fusion protein was identifie...

In Silico Cloning,Expression and Phylogenetic Analysis of Pig GATA-3 Gene

Pig GATA-3 cDNA was obtained by reverse transcription polymerase chain reaction （RT-PCR）, using in silico cloning strategy based on pig dbEST. The length of pig GATA-3 cDNA is 1,760 bp containing a 1,335 bp open reading frame （ORF）, which encodes a 444 amino acid protein. Semi-quantitative RT-PCR analysis of GATA-3 mRNA expression was done using the total RNAs from different normal tissues of a large white pig. The GATA-binding family of transcription factors comprised of a subgroup of DNA-binding proteins that both bound the consensus GATA motif and contained the class Ⅳ zinc finger motif. The molecular evolution tree was constructed based on the GATA-3 amino acid sequence and class Ⅳ zinc finger motif using mega 3.1. Phylogeny analysis of GATA factors isolated from vertebrates suggested that the six distinct vertebrate GATAs had descended from a common ancestral sequence, and the topology also suggested multiple modes of evolution including gene duplication and class Ⅳ zinc finger motif recombination. These data helped the authors in illuminating the pathways of divergent and convergent evolution of the GATA-binding family of transcription factors.

Modeling Rice rgMT as a Plant Metallothionein-Like Protein by the Distance Geometry and Homology Methods

Rice metallothionein-like protein (rgMT) shows characteristics of a three-section pattern composed of two highly conserved cysteine rich (CR) domains in the terminals and a spacer without cysteine (cys) residues in the center of the molecule. In this paper, the two CR domains and the spacer region were modeled by the distance geometry and homology methods separately. For the CR domains, twenty random models were generated for each cys combination based on the constraint conditions of CXC (C represents cys, X represents any amino acid other than cys), and CXXC motifs and a metal-sulfur chelating cluster. Four models for the N-terminal and two for C-terminal CR domain containing metal chelating structures formed by different combinations of cys were selected from 900 possible conformations. The GOR method was used to predict the secondary structure of the spacer region and its model was built by the homology method. After three parts of the protein were modeled, they were connected to form a three-dimensional structure model of rgMT. The whole conformation showed that rgMT could form two independent metal-sulfur chelating structures connected by a spacer peptide, without a structural or energy barrier for them to form two independent metal-chelating clusters just as mammalian metallothionein (MT) proteins. As all plant metallothionein-like (MT-L) proteins have the same primary structural characteristic, two CR domains connected by a spacer region, and many have the same cys arrangement pattern as rgMT, the three-dimensional structure model of rgMT will provide an important reference for the structural study of other plant MT-L proteins.

Function of nonstructural 5A protein of genotype 2a in replication and infection of HCV with gene substitution

AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR)method and the restriction enzyme reaction.In vitro RNA transcripts of chimera,prototype J6JFH and negative control J6JFH1(GND)were prepared and transfected into Huh-7.5 cells with liposomes.Immunofluorescence assay(IFA),fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.RESULTS:The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point(2.58 ±5.97×106 vs 4.27±1.72×104,P=0.032).The maximal level of HCV RNA in chimera was 5.6±1.8× 104 GE/μg RNA at day 34 after transfection,while the wild type reached a peak level at day 13 which was 126 folds higher(70.65±14.11×105 vs 0.56±0.90 ×105,P=0.028).HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level.Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles,ranging from 10±5 ffu/mL to 78.3±23.6 ffu/mL,while that of FL-J6JFH1 ranged from 5.8±1.5×102 ffu/mL to 2.5±1.4×104 ffu/mL.CONCLUSION:JFH1 NS5A might play an important role in the robust replication of J6JFH1.The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.

RNA干扰大鼠H9c2心肌细胞Hsc70基因条件筛选

为了研究H9c2心肌细胞RNA干扰的转染条件,筛选结构型热休克蛋白70(Hsc70)干扰序列,采用流式细胞仪和Western blot方法,研究不同剂量比例荧光标记的siRNA(FAM-siRNA)和不同浓度脂质体2000(Lipofectamine 2000)对转染效率的影响,进一步筛选Hsc70 siRNA的最佳沉默序列。结果显示:流式细胞仪试验得出的最佳转染条件为80 nmol/L FAM-siRNA+1.5μL Lipofectamine 2000;Western blot结果表明,Oligo 2、Oligo 3和Oligo 4组中的Hsc70蛋白表达极显著降低(P<0.01),其中Oligo 3组的沉默效率最高;转染后48 h收获细胞为最佳转染时间。结果表明:80 nmol/L FAM-siRNA+1.5μL Lipofectamine 2000为最佳转染条件,以Oligo 3为最佳RNA干扰靶基因。

Effect of Lingguizhugan decoction on myocardial Nuclear factor kappa B protein expression in rats with chronic heart failure

OBJECTIVE: To investigate the effect of Lingguizhugan decoction (LGZGD) on changes of cardiac structure and function, and its putative mechanism of action, by investigating mRNA and protein expression of myocardial nuclear factor kappa B(NF-κB), and the plasma content of NF-κB in rats with chronic heart failure.METHODS: The chronic heart failure (CHF) model in rats was induced by coronary artery ligation.Sham operation was performed in control rats. Six weeks after the procedure, rats were randomly classified into the various treatment groups: model CHF, Captopril (4.4 mg/kg), low LGZGD dose (2.1 g/kg), medium LGZGD dose (4.2 g/kg), and high LG-ZGD dose (8.4 g/kg). Treatments continued for 4 consecutive weeks. Changes of hemodynamic indices were observed by the PowerLab data acquisition and analysis system. Morphological changes of myocardium were observed by hematoxylin and eosin staining, and Masson staining. The mRNA and protein expression of myocardial NF-κB were detected by reverse transcription-polymerase chain reaction and western blotting, respectively. The plasma content of NF-κB was detected by enzyme-linked immuno-sorbent assay.RESULTS: CHF rats showed significant dysfunction in hemodynamic indices and in cardiac structure.Compared with the sham operation group, mRNA expression of myocardial NF-κB and plasma content of NF-κB of the model group was significantly increased. All three doses of LGZGD, and Captopril,improved the hemodynamic dysfunction, and inhibited the change of cardiac structure while significantly improving the survival rate. Furthermore,compared with the model group, mRNA expression of myocardial NF-κB and plasma content of NF-κB were significantly reduced by all dosage groups of LGZGD as well as the\Captopril group.CONCLUSION: In CHF rats, LGZGD improves changes of cardiac structure and function via its inhibition of NF-κB.

Phylogenetic analysis revealed the central roles of two African countries in the evolution and worldwide spread of Zika virus

Recent outbreaks of Zika virus （ZIKV） infections in Oceania＇s islands and the Americas were characterized by high numbers of cases and the spread of the virus to new areas. To better understand the origin of ZIKV, its epidemic history was reviewed. Although the available records and information are limited, two major genetic lineages of ZIKV were identified in previous studies. However, in this study, three lineages were identified based on a phylogenetic analysis of all virus sequences from GenBank, including those of the envelope protein （E） and non-structural protein 5 （NS5） coding regions. The spatial and temporal distributions of the three identified ZIKV lineages and the recombination events and mechanisms underlying their divergence and evolution were further elaborated. The potential migration pathway of ZIKV was also characterized. Our findings revealed the central roles of two African countries, Senegal and Cote d＇lvoire, in ZIKV evolution and genotypic divergence. Furthermore, our results suggested that the outbreaks in Asia and the Pacific islands originated from Africa. The results provide insights into the geographic origins of ZlKV outbreaks and the spread of the virus, and also contribute to a better understanding of ZlKV evolution, which is important for the prevention and control of ZlKV infections.

Racemic X-ray structure of L-type calcium channel antagonist Calciseptine prepared by total chemical synthesis

Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by using hydrazide based native chemical ligation. The crystal structure of Calciseptine was determined by racemic protein crystallography technique. Compared to the structure of its homologous family protein, we found that Calciseptine is adopting a typical three-finger structure.

Structural and biochemical insights into the homotypic PB1-PB1 complex between PKCζ and p62

The atypical PKC isoforms （ζ and t） play essential roles in regulating various cellular processes. Both the hetero-interaction between PKCζand p62 through their N-terminal PB 1 domains and the homo-oligomerization of p62 via its PB 1 domain are critical for the activation of NF-r.B signaling; however, the molecular mechanisms concerning the formation and regulation of these homotypic complexes remain unclear. Here we determined the crystal structure of PKCζ-PB 1 in complex with a mono- meric p62-PB 1 mutant, where the massive electrostatic interactions between the acidic OPCA motif of PKCζ-PB 1 and the basic surface of p62-PB 1, as well as additional hydrogen bonds, ensure the formation of a stable and specific complex. The PKCζ-p62 interaction is interfered with the modification of a specific Cys of PKCζ by the antiarthritis drug aurothiomalate, though all four cysteine residues in the PKCζ-PB 1 domain can be modified in in vitro assay. In addition, detailed structural and biochemical analyses demonstrate that the PB 1 domains of aPKCs belong to the type I group, which can depolymerize the high-molecular-weight p62 aggregates into homo-oligomers of lower order. These data together unravel the molecular mecha- nisms of the homo- or hetero-interactions between p62 and PKCζ and provide the basis for designing inhibitors of NF-r,.B sig- naling.

Effect of conventional medical treatment plus Qigong exercise on type 2 diabetes mellitus in Chinese patients： A Meta-analysis

OBJECTIVE: To systematically evaluate the effect of conventional medical treatment plus Qigong exercise on type 2 diabetes mellitus(T2 DM) in Chinese patients.METHODS: A literature search was conducted in Pub Med, Medline, Embase, China National Knowledge Infrastructure Database, Wanfang and China Science and Technology Journal Database until on January 2017. In this Meta-analysis, enrolled were the studies, including experimental group(with conventional medical treatment plus Qigong therapy) and control group(with conventional medicaltreatment plus diet control or other exercises).Then, the standardized mean differences(SMDs) of fasting blood-glucose(FBG), 2-hplasmaglucose(2 h PG), hemoglobin Alc(Hb Alc), triglyceride(TG),total cholesterol(TC), high-density lipoprotein(HDL) and low density lipoprotein(LDL), as well as their 95% confidence interval(CIs) were calculated to evaluate the effect of Qigong on patients with T2 DM by Rev Man 5.2 and Stata 11.0 software.RESULTS: Totally 834 cases of T2 DM from 11 studies were included in this review. The pooled SMDs with its 95% CIs of FBG, 2 h PG, Hb Alc, TG,TC, HDL and LDL were-0.70(-0.93,-0.47),-0.66(-1.11,-0.21),-0.73(-0.96,-0.50),-1.05(-1.67,-0.43),-0.42(-1.12, 0.28), 0.69(0.19, 1.19), and-0.26(-0.69, 0.18), respectively.The pooled data showed significantly difference between Qigong and the levels of FBG, 2 h PG, Hb A1 c,TG, and HDL in patients with T2 DM(P < 0.05).CONCLUSION: Combining with conventional medical treatment, Qigong exercise might have significant effect on T2 DM in Chinese patients.

Comparison of reduced point charge models of proteins: Molecular Dynamics simulations of Ubiquitin

Reduced point charge models of amino acids are used to model Ubiquitin （PDB： 1UBQ）. They are designed （i） from local ex- tremum positions in charge density （CD） distribution functions built from the Poisson equation applied to smoothed molecular electrostatic potential functions, or （ii） from local maximum positions in promolecular electron density distribution （ED） func- tions. Charge values are fitted versus all-atom Amber99 molecular electrostatic potentials. The program GROMACS is used to generate molecular dynamics trajectories of the protein, under various implementation schemes, solvation, and temperature conditions. Point charges that are not located on atoms are considered as virtual sites with a null mass and radius. The results illustrate that secondary structure is best preserved with the CD-based model at low temperatures and in vacuum. This indi- cates that local potential energy wells are consistent with the all-atom model. However, at room temperature, the structure is best conserved when point charges are forced to be located on atoms, due to a better description of the Coulomb l-4 energy terms. The ED-based model, generated at a lower resolution, led to the largest discrepancies versus the all-atom case. The CD-based model allows the formation of protein-water H-bonds with geometrical properties similar to the all-atom ones. Con- trarily, intra-molecular H-bonds are not well described. Structural, thermodynamical, and dynamical properties of proteins modelled with reduced point charge models are also significantly affected by the choice of the solvent force field.

Heterologous interactions between NS1 proteins from different influenza A virus subtypes/strains

Non-structural protein 1 （NS1） of the influenza virus plays a crucial role in modulating the host immune response and facili- tating virus replication. The formation of a homodimer or an oligomer is necessary for NSI to exert its function efficiently. In the present study, the NS 1 protein from the A/Shantou/602/06（H3N2） virus （herein abbreviated as NS32） was found to interact with NS1 from A/Shantou/169/O6（H1N1）, A/Chicken/Guangdong/1/05（HSN1） and A/Quail/Hong Kong/G1/97（H9N2） （abbre- viated as NS11, NS51 and NS92, respectively） viruses, although NS32 shares 17.4%-20.9% sequence diversity with NS11, NS51 and NS92. This indicates that the heterologous interactions between NS1 proteins from different influenza A virus sub- types/strains may be a common event during co-infection.