蛋白质分析和蛋白质组学0

蛋白质分析和蛋白质组学,基础学院 病理生理学教研室,引言,从四个视角看蛋白质,蛋白质组学,分析细胞通路的生物信息学方法,Protein analysis and proteomics,Individual proteins Perspective 1: Protein families (domains and motifs) Perspective 2: Physical properties (3D structure) Perspective 3: Localization Perspective 4: Function,protein,Page 389,RNA,DNA,protein,1 Protein families,4 Protein function,2 Physical properties,Page 389,3 Protein localization,Gene ontology (GO):-cellular component-biological process-molecular function,从四个视角看蛋白质,蛋白质家族,蛋白质的物理性质,蛋白质的细胞定位,蛋白质的功能,Perspective 1: Protein domains and motifs,Page 389,从四个视角看蛋白质,蛋白质家族,蛋白质的模块性质,具有同源序列的蛋白质，两条序列存在显著相似的区域。,具有序列相似性或显著结构特征的区域,签名,结构域,模块,模块元件,折叠子,模体,模式,重复,Definitions,Signature: a protein category such as a domain or motif,结构域,蛋白质中能折叠成特定三维结构的一段区域，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。,Definitions,Signature: 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 domainMotif (or fingerprint): a short, conserved region of a protein typically 10 to 20 contiguous amino acid residues,Page 390,John Reed 等人于2001发现的新基因Bcl-GL。 B。 该基因BH3 domains 与Bcl-2 家族其他蛋白质的比对分析。 相同与相似的残基分别用黑色和灰色出题粗体字母标示 C, 上述蛋白质BH2 domains的比对分析,EBV 编码的BALF1 ，BHRF1 蛋白质与 Kaposis sarcoma-associated herpesvirus （HHV8）ORF16 (KSHVorf16) 与细胞基因bcl-x ，bcl-2的序列相似性。一致性的区域为黑色，保守区域为灰色。,15 most common domains (human),Zn finger, C2H2 type1093 proteinsImmunoglobulin1032EGF-like471Zn-finger, RING458Homeobox417Pleckstrin-like405RNA-binding region RNP-1400SH3394Calcium-binding EF-hand392Fibronectin, type III300PDZ/DHR/GLGF280Small GTP-binding protein 261BTB/POZ236bHLH226Cadherin226,Page 391,Source: Integr8 at EBI website,Definition of a domain,According to InterPro at EBI (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 structure content and a hydrophobic core.Homologous domains with common functions usuallyshow sequence similarities.,Page 390,Varieties of protein domains,Page 393,Extending along the length of a protein,Occupying a subset of a protein sequence,Occurring one or more times,Figure 4 Stereoscopic view of the Ins(1,4,5)P3-binding site of the PLCdelta1 PH domain,Biochemical Journal Biochem. J. (2000) 350, 1-18,Model of phosphoinositide binding and phosphorylation 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 associated 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 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, 2000,Example of a protein with domains: Methyl CpG binding protein 2 (MeCP2),MBD,Page 393,TRD,The protein includes a methylated DNA binding domain(MBD) and a transcriptional repression domain (TRD).MeCP2 is a transcriptional repressor.Mutations in the gene encoding MeCP2 cause RettSyndrome, a neurological disorder affecting girlsprimarily.,Page 393,Result of an MeCP2 blastp search:A methyl-binding domain shared by several proteins,domain,Definition of a motif,A motif (or fingerprint) 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 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,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，通常决定一个蛋白质家族。,长度一般是10-20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 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,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 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 proteins,Page 397,蛋白质的物理性质,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,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,从四个视角看蛋白质,蛋白质的细胞定位,蛋白质的功能,从四个视角看蛋白质,蛋白质的细胞定位,从四个视角看蛋白质,蛋白质的功能,蛋白质组学,四种进行高通量分析的手段,二维凝胶电泳,亲和层析串联质谱,酵母双杂交系统,基于基因组DNA序列分析（Rosetta 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。,蛋白质是两性离子,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 one (D) are presented. Proteins (100g loaded) were separated over pH range 58 (17cm 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,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,Bait蛋白,导入酵母细胞,电泳纯化蛋白复合物,切割含有蛋白质条带的凝胶,质谱分析，确定蛋白质序列,四