蛋白质分析和蛋白质组学(0)

1、蛋白质分析和蛋白质组学蛋白质分析和蛋白质组学基础学院基础学院 病理生理学教研室病理生理学教研室u引言引言u从四个视角看蛋白质从四个视角看蛋白质u蛋白质组学蛋白质组学u分析细胞通路的生物信息学方法分析细胞通路的生物信息学方法Protein analysis and proteomicsIndividual proteins Perspective 1: Protein families (domains and motifs) Perspective 2: Physical properties (3D structure) Perspective 3: Localization Perspec

2、tive 4: FunctionproteinPage 389RNADNAprotein1 Protein families4 Protein function2 Physical propertiesPage 3893 Protein localizationGene ontology (GO):-cellular component-biological process-molecular functionu从四个视角看蛋白质从四个视角看蛋白质蛋白质家族蛋白质家族蛋白质的物理性质蛋白质的物理性质蛋白质的细胞定位蛋白质的细胞定位蛋白质的功能蛋白质的功能Perspective 1: Prote

3、in domains and motifsPage 389u从四个视角看蛋白质从四个视角看蛋白质蛋白质家族蛋白质家族蛋白质的模块性质蛋白质的模块性质具有同源序列的蛋白质，两条序列存在具有同源序列的蛋白质，两条序列存在显著相似的区域。显著相似的区域。具有序列相似性或显著结构特征的区域具有序列相似性或显著结构特征的区域签名签名结构域结构域模块模块模块元件模块元件折叠子折叠子 模体模体模式模式重复重复DefinitionsSignature: a protein category such as a domain or motif结构域结构域蛋白质中能折叠成特定三维结构的一段区域，蛋白质中能折叠成特

4、定三维结构的一段区域，也称为模块也称为模块具有相同结构域的蛋白被称为一个蛋白质家族。具有相同结构域的蛋白被称为一个蛋白质家族。DefinitionsSignature: a protein category such as a domain or motifDomain: a region of a protein that can adopt a 3D structure a fold a family is a group of proteins that share a domain examples: zinc finger domain immunoglobulin domainMo

5、tif (or fingerprint): a short, conserved region of a protein typically 10 to 20 contiguous amino acid residuesPage 390John Reed John Reed 等人于等人于20012001发现的新基因发现的新基因Bcl-GLBcl-GL。 B B。 该基该基因因BH3 domains BH3 domains 与与Bcl-2 Bcl-2 家族其他蛋白质的比对分析。家族其他蛋白质的比对分析。 相相同与相似的残基分别用黑色和灰色出题粗体字母标示同与相似的残基分别用黑色和灰色出题粗体字母

6、标示 C, C, 上述蛋白质上述蛋白质BH2 domainsBH2 domains的比对分析的比对分析EBV 编码的编码的BALF1 ，BHRF1 蛋白质与蛋白质与 Kaposis sarcoma-associated herpesvirus （HHV8）ORF16 (KSHVorf16) 与与细胞基因细胞基因bcl-x ，bcl-2的序列相似性。一致性的区域为黑色，的序列相似性。一致性的区域为黑色，保守区域为灰色。保守区域为灰色。15 most common domains (human)Zn finger, C2H2 type1093 proteinsImmunoglobulin1032E

7、GF-like471Zn-finger, RING458Homeobox417Pleckstrin-like405RNA-binding region RNP-1400SH3394Calcium-binding EF-hand392Fibronectin, type III300PDZ/DHR/GLGF280Small GTP-binding protein 261BTB/POZ236bHLH226Cadherin226Page 391Source: Integr8 at EBI websiteDefinition of a domainAccording to InterPro at EBI

8、 (http:/www.ebi.ac.uk/interpro/):A domain is an independent structural unit, found aloneor in conjunction with other domains or repeats.Domains are evolutionarily related. According to SMART (http:/smart.embl-heidelberg.de):A domain is a conserved structural entity with distinctivesecondary structur

9、e content and a hydrophobic core.Homologous domains with common functions usuallyshow sequence similarities.Page 390Varieties of protein domainsPage 393Extending along the length of a proteinOccupying a subset of a protein sequenceOccurring one or more timesModel of phosphoinositide binding and phos

10、phorylation in activation of Vav GEF activity. The inactive Vav molecules is bound to PI-4,5-P2 (PIP2) and tyrosine 174 (Y) is not phosphorylated. In this inactive state, the PH domain interacts with DH domain sequences to obscure the Rac-binding site. Upon PI 3-kinase activation, Vav becomes associ

11、ated with PI-3,4,5-P3 (PIP3) and a conformational change in the PH domain occurs that reduces its affinity for the DH domain. The altered conformation allows the Lck kinase to phosphorylate a tyrosine residue (circled P) N-terminal to the DH domain. This further reduces the affinity of the PH domain

12、 for the DH domain, and then Rac protein can bind to the DH domain. J Biol Chem, Vol. 275, Issue 20, 15074-15081, May 19, 2000Example of a protein with domains: Methyl CpG binding protein 2 (MeCP2)MBDPage 393TRDThe protein includes a methylated DNA binding domain(MBD) and a transcriptional repressio

13、n domain (TRD).MeCP2 is a transcriptional repressor.Mutations in the gene encoding MeCP2 cause RettSyndrome, a neurological disorder affecting girlsprimarily. Page 393Result of an MeCP2 blastp search:A methyl-binding domain shared by several proteinsdomainDefinition of a motifA motif (or fingerprint

14、) is a short, conserved region of a protein. Its size is often 10 to 20 amino acids.Simple motifs include transmembrane domains andphosphorylation sites. These do not imply homologywhen found in a group of proteins.PROSITE (/prosite) is a dictionary of motifs (there are currently 1600

15、entries). In PROSITE,a pattern is a qualitative motif description (a proteineither matches a pattern, or not). In contrast, a profileis a quantitative motif description. We will encounterprofiles in Pfam, ProDom, SMART, and other databases.Page 394模体模体蛋白质序列中较短的保守区域蛋白质序列中较短的保守区域按一定模式排列的氨基酸残基，通常决定按一定模

16、式排列的氨基酸残基，通常决定一个蛋白质家族。一个蛋白质家族。长度一般是长度一般是10-2010-20个氨基酸残基。个氨基酸残基。FIG. 1. Composition of the NPXY/NXXY motif database and peptide synthesis. Origin of all 126 NXXY motifs identified by surveying human protein sequencesclassified as receptors. Twenty percent of the sites were derived from RTKs, several

17、 of which containmultiple motifs. Mol Cell Biol. 2006 Nov;26(22):8461-74. Epub 2006 Sep 18 蛋白质模式：模体或蛋白质的指纹特征蛋白质模式：模体或蛋白质的指纹特征Summary of Perspective 1: Protein domains and motifs A signature is a protein category such as a domain or motif.You can learn about domains at Integr8, and at databases such

18、as InterPro and Pfam. A motif (or fingerprint) is a short, conserved sequence. You can study motifs at Prosite at ExPASy. Perspective 2: Physical properties of proteinsPage 397蛋白质的物理性质蛋白质的物理性质可能由翻译后的修饰造成。可能由翻译后的修饰造成。有些修饰可以让疏水基团与蛋白质共价结合而有些修饰可以让疏水基团与蛋白质共价结合而使蛋白质插入脂质双分子层使蛋白质插入脂质双分子层评价和预测蛋白质物理性质的网页评价和预测

19、蛋白质物理性质的网页预测分子量，等电点，氨基酸组成成分，预测分子量，等电点，氨基酸组成成分，糖基化位点，磷酸化位点和硫化位置糖基化位点，磷酸化位点和硫化位置u从四个视角看蛋白质从四个视角看蛋白质蛋白质的细胞定位蛋白质的细胞定位蛋白质的功能蛋白质的功能u从四个视角看蛋白质从四个视角看蛋白质蛋白质的细胞定位蛋白质的细胞定位u从四个视角看蛋白质从四个视角看蛋白质蛋白质的功能蛋白质的功能u蛋白质组学蛋白质组学四种进行高通量分析的手段四种进行高通量分析的手段二维凝胶电泳二维凝胶电泳亲和层析串联质谱亲和层析串联质谱酵母双杂交系统酵母双杂交系统基于基因组基于基因组DNADNA序列分析序列分析（Rosetta

20、 stoneRosetta stone）四种进行高通量分析的手段二维凝胶电泳二维：根据分子大小与迁移率的反比关系，将混合液二维：根据分子大小与迁移率的反比关系，将混合液中各个组分分开中各个组分分开一维：等电聚焦使蛋白质根据电荷初步分离。一维：等电聚焦使蛋白质根据电荷初步分离。蛋白质是两性离子蛋白质是两性离子被分离成分用来直接测序被分离成分用来直接测序Fig. 1. Real gels of protein extracts obtained using the TCAacetone (A), phenol (B), and TCAacetonephenol (C), and a master

21、one (D) are presented. Proteins (100 g loaded) were separated over pH range 58 (17 cm strips) and 12% SDS-polyacrylamide gel. The gel was stained with Sypro Ruby. Numbered spots corresponded to those showing qualitative or statistically significant quantitative differences. 四种进行高通量分析的手段四种进行高通量分析的手段亲和层析串联质谱亲和层析串联质谱确定酵母细胞中存在蛋白复合物的方法确定酵母细胞中存在蛋白复合物的方法Bait蛋白蛋白导入酵母细胞导入酵母细胞电泳纯化蛋白复合物电泳纯化蛋白复合物切割含有蛋白质条带的凝胶切割含有蛋白质条带的凝胶质谱分析，确定蛋白质序列质谱分析，确定蛋白质序列四种