The structural analysis of protein sequences based on the quasi-amino acids code

Proteomics is the study of proteins and their interactions in a cell. With the successful completion of the Human Cenome Project, it comes the postgenome era when the proteomics technology is emerging. This paper studies protein molecule from the algebraic point of view. The algebraic system （∑, ＋, ＊） is introduced, where ∑ is the set of 64 codons. According to the characteristics of （∑, ＋, ＊）, a novel quasi-amino acids code classification method is introduced and the corresponding algebraic operation table over the set ZU of the 16 kinds of quasi-amino acids is established. The internal relation is revealed about quasi-amino acids. The results show that there exist some very close correlations between the properties of the quasi-amino acids and the codon. All these correlation relationships may play an important part in establishing the logic relationship between codons and the quasi-amino acids during the course of life origination. According to Ma F et al （2003 J. Anhui Agricultural University 30 439）, the corresponding relation and the excellent properties about amino acids code are very difficult to observe. The present paper shows that （ZU, ＋,×） is a field. Furthermore, the operational results display that the eodon tga has different property from other stop codons. In fact, in the mitochondrion from human and ox genomic codon, tga is just tryptophane, is not the stop codon like in other genetic code, it is the case of the Chen W C et al （2002 Acta Biophysiea Siniea 18（1） 87）. The present theory avoids some inexplicable events of the 20 kinds of amino acids code, in other words it solves the problem of ＇the 64 codon assignments of mRNA to amino acids is probably completely wrong＇ proposed by Yang （2006 Progress in Modern Biomedicine 6 3）.

Favorable and unfavorable amino acid residues in water-soluble and transmembrane proteins

We analyzed the amino acid residues present in the water-soluble and transmembrane proteins of 6 thermophilic and 6 mesophilic species of the domains Archaea and Eubacteria, and characterized them as favorable or unfavorable. The characterization was performed by comparing the observed number of each amino acid residue to the expected number calculated from the percentage of nucleotides present in each gene. Amino acids that were more or less abundant than expected were considered as favorable or unfavorable, respectively. Comparisons of amino acid compositions indicated that the water-soluble proteins were rich in charged residues such as Glu, Asp, Lys, and His, whereas hydrophobic residues such as Trp, Phe, and Leu were abundant in transmembrane proteins. Interestingly, our results found that although the Trp residue was abundant in transmembrane proteins, it was not defined as favorable by our calculations, indicating that increased numbers of a particular amino acid does not necessary indicate it is a favorable residue. Amino acids with high G + C content such as Ala, Gly, and Pro were frequently observed as favorable in species with low G + C content. Comparatively, amino acids with low G + C content such as Phe, Tyr, Lys, Ile, and Met were frequently observed as favorable in species with high G + C content. These are the examples to increase the supply of amino acids than expected. Amino acids with neutral G + C content, i.e., Glu and Asp were favorable in water-soluble proteins from all species analyzed, and Cys was unfavorable both in water-soluble and transmembrane proteins. These results indicate that amino acid compositions are essentially determined by the nucleotide sequence of the genes, and the amino acid content is altered by a deviation from expectation.

Differences in Amino Acid Composition between <i>α</i>and <i>β</i>Structural Classes of Proteins

The amino acid composition of α and β structural class of proteins from five species, Escherichia coli, Thermotoga maritima, Thermus thermophilus, yeast, and humans were investigated. Amino acid residues of proteins were classified into interior or surface residues based on the relative accessible surface area. The hydrophobic Leu, Ala, Val, and Ile residues were rich in interior residues, and hydrophilic Glu, Lys, Asp, and Arg were rich in surface residues both in α and β proteins. The amino acid composition of α proteins was different from that of β proteins in five species, and the difference was derived from the different contents of their interior residues between α and β proteins. α-helix content of α proteins was rich in interior residues than surface ones. Similarly, β-sheet content of β proteins was rich in interior residues than surface ones. The content of Leu residues was very high, approximately 20%, in interior residues of α proteins. This result suggested that the Leu residue plays an important role in the folding of α proteins.

Analysis of the N Protein Sequence Variability in 13 Isolated PRRSV Strains from China

Object: To analyze porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 infection cases via the N protein gene and its encoded amino acid sequence and to provide a theoretical basis for the epidemiological study, prevention and control of porcine reproductive and respiratory syndrome (PRRS). Methods: In clinically suspected PRRSV infections, viruses were isolated by extracting viral nucleic acid and amplifying the N protein gene by RT-PCR. Then, the product was purified and sequenced to acquire the whole gene sequence of the N protein and its encoded amino acid sequence. DNASTAR software was used to analyze the homology, the genetic evolution and the derivation of the variability of amino acids of the N protein gene from 13 PRRSV strains and classical domestic and foreign strains. Results: Among the thirteen strains of PRRSV isolated from this study, ten strains had the greatest homology with the JXA1 strain (98.9% - 100%), and they belonged to the sublineage 8.7. The remaining three strains had the greatest homology with the NADC30 strain (95.4% - 97.1%), and they belonged to lineage one. The analysis of the variability of N protein amino acids showed that there were high frequency mutations in the five loci of 13 isolated strains of PRRSV as follows: 15th amino acid (10/13), 46th amino acid (11/13), 91st amino acid (10/13), 109th amino acid (10/13), and 117th amino acid (10/13). Conclusion: In recent years, sublineage 8.7 was the dominant pedigree in field PRRSV epidemic strains in China with lineage one occupying a certain proportion of the field. Four high frequency mutations existed in N protein antigen epitopes of isolated strains from the region. The nuclear localization signal (NLS) structure, specifically the 46th amino acid residue of the N protein, was mutated and genetically stable.

非洲猪瘟病毒p30蛋白氨基酸序列分段表达及反应原性分析

为比较非洲猪瘟病毒(African swine fever virus,ASFV)p30蛋白氨基酸序列中不同片段的反应原性,探究p30蛋白氨基酸序列中可能的抗原优势片段。本研究通过PCR、克隆技术将p30蛋白氨基酸序列中不同片段的编码基因分别插入表达质粒pET32a,经IPTG诱导、Ni柱纯化、透析后,获得p30蛋白氨基酸序列中8~101 aa片段(P-1#)、58~101 aa片段(P-2#)、101~158 aa片段(P-3#)、8~194 aa片段(P-4#),用酶联免疫吸附试验(ELISA)分析p30蛋白氨基酸序列中不同片段与p30蛋白免疫兔血清和ASFV阳性猪血清的反应原性。结果显示,上述片段均获得诱导表达及纯化,表达形式有可溶性表达(P-1#、P-3#)和包涵体表达(P-2#、P-4#)。各片段以1.0 mg/L包被时,p30免疫兔血清与p30蛋白氨基酸序列中4种片段均能发生免疫反应,ASFV阳性猪血清与P-4#、P-1#反应较佳,与P-3#反应中等,与P-2#反应最弱。综上,p30蛋白氨基酸序列中不同片段反应原性的差异为认识p30抗原优势区域提供了数据,这将有助于非洲猪瘟血清学诊断抗原的科学筛选。

蛋白质分子表面氨基酸突变提高植酸酶Yi APPA的活性和热稳定性

为了提高植酸酶的活性和热稳定性,增加其在食品加工领域的应用潜力,对植酸酶Yi APPA进行同源模建,结合蛋白质分子表面氨基酸突变策略,选择位于分子表面的赖氨酸和甘氨酸进行定点突变,构建单位点突变体。通过活性和热稳定性筛选,获得活性和热稳定性显著提高的突变体K216R以及热稳定性提高的突变体K189R。通过有益突变位点叠加策略,进一步构建并表征组合突变体K189R/K216R的酶活力及热稳定性。结果表明:与Yi APPA相比,K189R/K216R于80℃半衰期由14.81 min延长至23.35 min,半失活温度由55.12℃提升至62.44℃,热解折叠温度由48.36℃提升至53.18℃。并且K189R/K216R于37℃、pH 4.5的酶活力由3959.98 U/mg提高至4469.13 U/mg。分子结构建模和分子动力学模拟的结果显示:K189R/K216R中引入了新的氢键,能够提高酶部分结构单元的稳定性,使其热稳定性得到提高;同时K189R/K216R的催化口袋体积增大是其活性提高的主要原因。本研究通过蛋白质分子表面氨基酸突变策略可有效提高植酸酶Yi APPA的活性和热稳定性,为植酸酶及其他类型酶的分子改造提供参考依据。

氨基酸配方替代喂养对重度牛奶蛋白过敏婴儿肠道菌群特征的影响

目的探讨氨基酸配方(AAF)替代喂养对重度牛奶蛋白过敏(CMPA)婴儿肠道菌群特征的影响。方法纳入23例确诊为重度CMPA且人工喂养的≤3月龄婴儿,采用同一品牌AAF进行喂养干预。干预前及干预3个月后,收集所有婴儿的粪便样本,通过16S rRNA高通量测序及生物信息学分析评估肠道菌群的特征变化。结果干预前后,重度CMPA婴儿肠道菌群Chao1指数、Shannon指数、observed species指数、Simpson指数差异具有统计学意义(P<0.05),Beta多样性具有差异,且组间差异大于组内差异。门水平的相对丰度分析及LEfSe分析结果均显示,AAF干预后重度CMPA婴儿肠道中厚壁菌门、拟杆菌门显著富集。属水平的相对丰度分析结果显示,AAF干预后重度CMPA婴儿肠道中拟杆菌属、粪杆菌属、双歧杆菌属的相对丰度较干预前明显升高,弓形菌属、克雷伯菌属、梭菌属的相对丰度较干预前降低,LEfSe分析结果显示拟杆菌属、粪杆菌属、弓形菌属、克雷伯菌属为在丰度上有显著差异的标志物种。在种水平上相对丰度排名前10的菌群中,产气荚膜梭菌、产酸拟杆菌、丁酸梭菌、格氏乳杆菌、罗伊氏乳杆菌的相对丰度显著升高。结论AAF替代喂养可使得重度CMPA患儿肠道菌群多样性增加,拟杆菌、厚壁菌等的丰度增加。

青花菜多聚半乳糖醛酸酶基因BoPGX3的克隆及盐胁迫下的表达特征分析

多聚半乳糖醛酸酶(PG)可降解细胞壁,从而降低植物抵抗能力。为了解青花菜PG基因响应盐胁迫的分子机制,以青花菜自交系‘WN12-95B’为试验材料,提取青花菜的总RNA并合成cDNA,克隆PG基因,并检测其在盐胁迫下的表达特征。结果表明:青花菜BoPGX3基因序列全长1476 bp,推导编码491个氨基酸,相对分子量为53.44 kD,等电点为5.84。系统进化分析表明,青花菜BoPGX3与羽衣甘蓝、甘蓝、白菜、萝卜和花椰菜的PG基因亲缘关系较近。荧光定量PCR结果显示,BoPGX3基因受盐胁迫诱导表达,处理3 h后相对表达量达到初始表达量的2.9倍,处理12 h时相对表达量下降至初始表达量的1.4倍。综上,青花菜BoPGX3基因参与了盐胁迫的响应,并可能在此过程中发挥着重要的作用。

高原鼢鼠和高原鼠兔CYP3A25基因的分子表征及功能分析

【目的】探究高原鼢鼠(Eospalax baileyi)和高原鼠兔(Ochotona curzoniae)CYP3A25基因的分子结构及蛋白质的三维构象差异。【方法】检测高原鼢鼠和高原鼠兔CYP3A25基因外显子的核苷酸序列情况,并结合其他7种啮齿动物分析其氨基酸序列和蛋白质空间构象差异。【结果】在PCR结果检测中成功获得高原鼢鼠和高原鼠兔CYP3A25基因CDS区,长度均为1307 bp,并检测出基因序列差异及蛋白质空间构象差异。系统发育树构建发现高原鼢鼠与色列盲鼹鼠(Nannospalax galili)属于同一分支,高原鼠兔更趋同于智人(Homo sapiens)。对CYP3A25基因序列差异介导蛋白质空间构象改变分析后,发现高原鼢鼠和高原鼠兔相较小家鼠都有3个氨基酸序列差异导致的蛋白质空间构象改变。【结论】研究表明不同物种CYP3A25基因存在较大的序列差异,并且这种序列差异影响了蛋白质的空间构象。说明不同物种在CYP3A25基因的分子表征和潜在的解毒代谢能力上存在较大差异。研究为理解不同物种的解毒代谢基因差异提供了理论依据。

Modeling and analysis of Schistosoma Argonaute protein molecular spatial conformation

Objective:To analyze the amino acid sequence composition,secondary structure,the spatial conformation of its domain and other characteristics of Argonaute protein.Methods:Bioinformatics tools and the internet server were used.Firstly,the amino acid sequence composition features of the Argonaute protein were analyzed,and the phylogenetic tree was constructed.Secondly,Argonaute protein's distribution of secondary structure and its physicochemical properties were predicted.Lastly,the protein functional expression form of the domain group was established through the Phyre-based analysis on the spatial conformation of Argonaute protein domains.Results:593 amino acids were encoded by Argonaute protein,the phylogenetic tree was constructed,and Argonaute protein's distribution of secondary structure and its physicochemical properties were obtained through analysis.In addition,the functional expression form which comprised the N-terminal PAZ domain and C-terminal Piwi domain for the Argonaute protein was obtained with Phyre.Conclusions:The information relationship between the structure and function of the Argonaute protein can be initially established with bioinformatics tools and the internet server,and this provides the theoretical basis for further clarifying the function of Schistosoma Argonaute protein.

Preparation of protein samples for gel electrophoresis by sequential extraction

Since preparation and solubilization of protein samples are crucial factors in proteome research,the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm.Bombyx mori.Two kinds of protein samples were obtained from the body wall using the method.Between the two types of samples only about 15% proteins were identical;the majority were different,indicating that more species of proteins could be obtained with the sequential extraction method;which will be useful for preparation of protein samples for proteome study.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide &quot;ALAHGVRAL (core 150-158)&quot;. The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

Proteolipid protein 1 gene sequencing of hereditary spastic paraplegia

PCR amplification and sequencing of whole blood DNA from an individual with hereditary spastic paraplegia, as well as family members, revealed a fragment of proteolipid protein 1 （PLP1） gene exon 1, which excluded the possibility of isomer 1 expression for this family. The fragment sequence of exon 3 and exon 5 was consistent with the proteolipid protein 1 sequence at NCBI. In the proband samples, a PLP1 point mutation in exon 4 was detected at the basic group of position 844, T→C, phenylalanine→leucine. In proband samples from a male cousin, the basic group at position 844 was C, but gene sequencing signals revealed mixed signals of T and C, indicating possible mutation at this locus. Results demonstrated that changes in PLP1 exon 4 amino acids were associated with onset of hereditary spastic paraplegia.

Combined molecular docking, homology modeling and DFTmethod for the modification of bovine serum albumin (BSA) toimprove fluorescence spectroscopy for phthalate acid esterschelated with BSA

While phthalate acid esters(PAEs)cannot fluoresce alone,they can be detected by fluorescence spectroscopy after chelation with bovine serum albumin(BSA).In this study,the types of amino acid residues at the active site of PAEs chelated with BSA were determined using molecular docking technology.A modification scheme of BSA with higher detection sensitivity fluorescence spectroscopy for PAEs was proposed based on the docking results and constructed for a novel BSA structure with a higher detection sensitivity of fluorescence spectroscopy using a homologous modeling method.Density functional theory(DFT)was employed to explore the influence before and after BSA modification on PAEs’detection through fluorescence spectroscopy.The results showed that the docking scores between BSAs and dimethyl phthalate(DMP),dibutyl phthalate(DBP)and di-n-octyl phthalate(DNOP)were increased up to 26.45%,16.82%and 16.30%,respectively,indicating that the active site modification of BSA could enhance the binding affinity between BSA and PAEs.The fluorescence intensity of PAEs chelated with modified BSAs were calculated.The fluorescence intensity of fluorescence spectroscopy for DMP,DBP and DNOP chelated with BSAs after modification was increased up to 2.8-,104.51-and 62.43-fold,respectively,which achieved the purpose of theoretically modifying BSA to improve the detection sensitivity of fluorescence spectroscopy for PAEs.

Review. Comparative structures and evolution of mammalian lipase I (LIPI) genes and proteins: A close relative of vertebrate phospholipase LIPH

Lipase I (enzyme name LIPI or LPDL) (gene name LIPI [human] or Lipi [mouse]) is a phospholipase which generates 2-acyl lysophosphatidic acid (LPA), a lipid mediator required for maintaining homeostasis of diverse biological functions and in activating cell surface recaptors. Bioinformatic methods were used to predict the amino acid sequences, secondary and tertiary structures and gene locations for LIPI genes and encoded proteins using data from several mammalian genome projects. LIPI is located on human chromosome 21 and is distinct from other phospholipase A1-like genes (LIPH and PS-PLA1). Mammalian LIPI genes contained 10 (human) or 11 (mouse) coding exons transcribed predominantly on the negative DNA strand. Mammalian LIPI protein subunits shared 61% - 99% sequence identities and exhibited sequence alignments and identities for key LIPI amino acid residues as well as extensive conservation of predicted secondary and tertiary structures with those previously reported for pancreatic lipase (PL), with “N-signal peptide”, “lipase” and “plat” structural domains. Comparative studies of mammalian LIPI sequences with LIPH, PS-PLA1 and pancreatic lipase (PL) confirmed predictions for LIPI N-terminal signal peptides (residues 1 - 15);predominantly conserved mammalian LIPI N-glycosylation sites (63NNSL and 396NISS for human LIPI);active site “triad” residues (Ser159;Asp183;His253);disulfide bond residues (238 - 251;275 - 286;289 - 297;436 - 455);and a 12 residue “active site lid”, which is shorter than for other lipases examined. Phylogenetic analyses supported a hypothesis that LIPI arose from a vertebrate LIPH gene duplication event within a mammalian common ancestral genome. In addition, LIPI, LIPH and PL-PLA1 genes were distinct from the vascular lipase (LIPG, LIPC and LPL) and pancreatic lipase (PL) gene families.

紫苏粕蛋白抗氧化活性肽的制备、分离纯化及序列鉴定

以紫苏粕粉为原料,使用碱性蛋白酶进行水解反应,制备抗氧化活性肽。采用超滤离心、凝胶过滤色谱和反相色谱等分离纯化手段对其富集。结果表明,相较其它3种蛋白酶,碱性蛋白酶Alcalase对紫苏粕蛋白的水解程度最高,约为(25.94±0.21)%;碱性蛋白酶作用紫苏蛋白后的酶解产物的抗氧化活性最好,对DPPH自由基清除率约为(91.01±0.73)%。酶解上清液经超滤离心分离后最小分子质量组分F1(小于3 ku)抗氧化活性最强,F1组分经Sephadex G-25凝胶过滤色谱分离后按照分子质量大小依次得到P1、P2、P33个组分,其中分子质量最小的P3组分抗氧化活性最高。P3组分经反相色谱分离所得4个组分中,最后被洗脱出来的P3-4组分疏水性最强且DPPH·自由基清除率最高,质量浓度为3μg/mL的P3-4溶液的DPPH·清除率为(58.8±0.78)%。通过LC-MS-MS鉴定,抗氧化活性肽P3-4组分为十二肽,其氨基酸序列为Lys-Leu-Lys-Asp-Ser-Phe-Glu-Arg-Gln-Gly-Met-Val,分子质量为1437.8 u。本研究结果为深入开发紫苏蛋白资源,研发紫苏抗氧化活性肽提供理论参考。

Genetic Characterization of Fusion Protein of Human Respiratory Syncytial Virus: Beijing

Objective Fusion protein is a subunit of the human respiratory syncytial virus(HRSV)and a potential vaccine candidate.Thus,a study on the genetic characteristics of F protein was considered important for further investigations in this field.The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide.Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing.Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs.Results Seventy-six samples(23.1%)were positive for HRSV.The percentage of cases in patients younger than 1year was 84.21%.Among the six Beijing isolates,four belonged to subgroup A,whose respective F genes shared97.0%-97.4%nucleotide sequence identity and 92.1%-93.0%amino acid sequence identity.The other two isolates belonged to subgroup B.Here,97.3%and 98.2%sequence identity were found at nucleotide and amino acid levels,respectively.Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other,but those two within subgroup B distributed in two distinct clusters.Subgroup A and B strains co-circulated,indicating that two different transmission chains occurred in Beijing from 2003-2004.

Purification and Partial Characterization of an Antibacterial Protein Tz1

A strain (T3) of Bacillus has been screened from paddy field. It secretes large amount of antibacterialproteins which show a strong inhibiting activity against several pathogens of rice. This paper presentsa systematic study of the inhibition spectrum and characteristics of T3 proteins. Total proteins wereprecipitated with ammonium sulfate at 70% saturation from cell-free culture. One of the proteins(Tzl) was purified from the crude extracts with Sephadex G-100, DEAE52 and FPLC Superose 12columns. A single band was demonstrated in both 15% SDS-PAGE and IEF, with an apparent MWof 6,9 kd and a pI of 7.8. Its amino acid composition was analyzed and part of its sequence,determined.

菌体蛋白作为饲料蛋白原料的研究进展

菌体蛋白饲料含有丰富的蛋白质,富含赖氨酸、蛋氨酸和色氨酸,饲喂动物后能提高饲料利用率和动物生产性能,增加养殖业经济效益。菌体蛋白的合成原料来源广泛,具有环境友好、富含粗蛋白和必需氨基酸等优点,对畜牧业的可持续发展和资源回收利用具有极其重要的意义。文章将菌体蛋白饲料进行分类并对其价值以及应用研究进行归纳总结,为菌体蛋白饲料的应用研究提供参考。

单宁酸对丝素蛋白二级结构的影响作用研究

蛋白质的二级结构是其发挥生物功能的重要基础结构,因此本文通过单宁酸和丝素蛋白反应,研究不同摩尔浓度的单宁酸对丝素蛋白二级结构的影响。使用紫外可见光吸收光谱(UV-Vis)、荧光发射光谱(FL)和傅里叶红外吸收光谱(FTIR)等表征技术对单宁酸与丝素蛋白的反应产物进行测试,结果表明单宁酸可以影响丝素蛋白的二级构象,并呈现一定的变化趋势。单宁酸摩尔浓度逐渐增加到20μmol/L时,α螺旋和无规卷曲结构逐渐增多,β折叠结构逐渐减少。单宁酸摩尔浓度继续上升,α螺旋和无规卷曲结构逐渐减少,β折叠结构逐渐增多。