生物大分子相互作用-蛋白质与

1、二、概论二、概论分子细胞生物学前沿进展与技术分子细胞生物学前沿进展与技术 生物大分子相互作用生物大分子相互作用Interaction between Biomacromolecules（interactome）The biomacromolecules in cell ProteinDNARNAOthers Why do we need to explore the interaction?By their friends, you shall know them!The interaction of biomacromolecules is the fundamental basis of

2、any cellular functionInteractionRNADNAothersProteinThe focuses:u Protein-protein interactionu Protein-DNA interactionu Protein-RNA interactionThe importance of protein-protein interactionProtein-protein interactions refer to the association of protein molecules and the study of these associations fr

3、om the perspective of biochemistry, signal transduction and networks.Protein-protein interactions are involved in almost every aspects of biological processes.Protein-protein interactions provide potential drug targets.The methods commonly used for studying protein-protein interaction Chemical cross

4、linking 化学交联化学交联 Pull-down 沉降实验沉降实验 Co-immunoprecipitation (Co-IP) 免疫共沉淀免疫共沉淀 Yeast Two-hybrid (Y2H) 酵母双杂交酵母双杂交 Fluorescence Resonance Energy Transfer (FRET) 荧光共荧光共振能量转移振能量转移 Docking 嵌合计算嵌合计算 Biacore (Surface Plasmon Resonance, SPR) 表面等离子共表面等离子共振振 Chemical CrosslinkingPrinciple: physically coupling

5、two proteins with a chemical crosslinkerAdvantage: in vivo Crosslinking reagents:formaldehydephoto-activated crosslinking reagents L-Photo-Leucine L-Photo-MethionineSubsequent analysis:SDS-PAGE, Western blotting, etc Pull-downGST pull-down, His pull-downWestern blot result of His pull-downExample: D

6、etecting the interaction between FS and ANG using His pull-down experimentHow to further improve theexperiment design?Co-Immunoprecipitation(Western Blot or MS)Example: BAG2 was identified by Co-IP as a CHIP-binding proteinCHIP: carboxyl terminus of Hsp70-interacting protein2005 JBC, 280: 3867338681

7、Yeast Two-hybridPrinciple: the modular design of eukaryotic transcription factors, which often have separate DNA-binding and transcriptional activation domains. Transcription factor: DB + AD DB: usually fuse to bait proteinAD: prey protein or a cDNA libray Basis of the Two-Hybrid System.(GIBCO BRL i

8、nstruction manual) Yeast Two-hybridYeast Two-hybrid a gold standard for detecting interactions in vivo advantages:in vivohigh-throughput screening disadvantages:yeast (cellular environment; protein modification)over-expressed proteinnot working with membrane proteinsneed to be confirmed by other met

9、hodsDevelopment of Two-hybrid Technique Promegas CheckMate system: works in mammalian cells, more likely to have the proper folding and post-translational modifications split-ubiquitin system: good for membrane proteins ubiquitin: could be expressed as an N-terminal half (Nub) + a C-terminal half (C

10、ub). The two halves retain their affinity for each other and spontaneously reassemble to form the so-called split-ubiquitin, but not if a point mutation is generated (NubG) Fluorescence Resonance Energy Transfer (FRET)FluorescenceGFP and other FPs (fluorescent proteins)FRET: principle FRET is a dist

11、ance-dependent physical process by which energy is transferred nonradiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) The donor emission spectrum must significantly overlap the excitation spectrum of the acceptor 供体发射波与受体供体发射波与受体激发波有重叠激发波有重叠 The donor

12、and acceptor fluorophores must be in favorable mutual orientation 合适的空间取向合适的空间取向 The distance between the donor and acceptor fluorophores must be less than 100 距离距离200,000文献挖掘高通量XML,ASN.1,BioPAX,CSV,SIF,XML否OntoglyphDIPDIP //19,34463,168文献挖掘MIF,XIN是PPI真实性评估IntAcIntAct t intac

13、t/site intact/site 56,071138,931文献挖掘PSI-MI是控制词汇，预测pull-down实验HPRDHPRD 25,20537,581文献挖掘PSI-MI否PhosphoMotif FinderMINTMINT ma2.it/mint/ma2.it/mint/28,186103,808文献挖掘PSI-MI,Osprey是HomoMINTMIPSMIPS 9001,800文献挖掘PSI-MI,XML否PEDANTThe usage of PPI resources Protein-protein interactions: binary interactions, molecular complexes The network of interacting proteins Pathways (functional) Evolution of protein-protein interactionThe possible interaction network of angiogenin and i