生物大分子相互作用

Chemical Crosslinking
Principle: physically coupling two proteins with a chemical crosslinker Advantage: in vivo Crosslinking reagents: 1. formaldehyde 2. photo-activated crosslinking reagents L-Photo-Leucine L-Photo-Methionine Subsequent analysis: SDS-PAGE and Western blotting
SPR: advantages & applications
优点： 优点：
无需对样品进行标记 实时监测 灵敏度高 快速
用处： 用处：
特异性—哪些分子发生了相互作用 特异性 哪些分子发生了相互作用? 哪些分子发生了相互作用 浓度—存在多少结合分子 存在多少结合分子? 浓度 存在多少结合分子 动力学—相互作用的速率 结合和解离的比例是多少? 相互作用的速率、 动力学 相互作用的速率、结合和解离的比例是多少 亲和性—相互作用的程度有多大 相互作用的程度有多大? 亲和性 相互作用的程度有多大 协同作用—是否存在任何异构效应 是否存在任何异构效应? 协同作用 是否存在任何异构效应 相互作用模式—结合模式与不同样品是否存在对应关系 结合模式与不同样品是否存在对应关系？ 相互作用模式 结合模式与不同样品是否存在对应关系？
2005 JBC, 280: 38673–38681
Yeast Two-hybrid
Principle: the modular design of eukaryotic transcription factors, which often have separate DNA-binding and transcriptional activation domains.
Development of Two-hybrid Technique
Promega's CheckMate system: works in mammalian cells, more likely to have the proper folding and posttranslational modifications split-ubiquitin system: good for membrane proteins
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The methods commonly used for studying protein-protein interaction
• Chemical crosslinking 化学交联 • Tagged Fusion Proteins (Pull-down) 沉降实验 • Co-immunoprecipitation (Co-IP) 免疫共沉淀 • Yeast Two-hybrid (Y2H) 酵母双杂交 • Fluorescence Resonace Energy Trasfer (FRET) 荧光共振 能量转移 • Docking 嵌合计算 • Biacore (Surface Plasmon Resonance, SPR) 表面等离子共 振
二、概论 1、分子细胞生物学前沿进展与技术 、
生物大分子相互作用
Interaction between Biomacromolecules
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The biomacromolecules in cell
Protein
DNA
RNA
Others
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The focuses:
Protein-protein interaction Protein-DNA interaction Protein-RNA interaction
The importance of protein-protein interaction
Protein-protein interactions refer to the association of protein molecules and the study of these associations from 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.
Co-Immunoprecipitation
(Western Blot or MS)
Example: BAG2 was identified by Co-IP as a CHIP-binding protein
CHIP: carboxyl terminus of Hsp70interacting protein
FRET: basic requirements
• The donor emission spectrum must significantly overlap the excitation spectrum of the acceptor 供体发射波与受体 激发波有重叠 • The donor and acceptor fluorophores must be in favorable mutual orientation 合适的空间取向 • The distance between the donor and acceptor fluorophores must be less 距离<100 Å than 100 Å 距离
Spectral imaging
Photobleaching
Photobleaching: an example
FRET: biosensor
e.g. Prostatic Specific Antigen (PSA): specifically recognize SSYYSG
CFP
G S G S G S S S Y Y S G G S G S G S
Pull-down
GST pull-down, His pull-down
Example: Detecting the interaction between FS and ANG using His pull-down experiment
Western blot result of His pull-down
The interaction of biomacromolecules is the fundamental basis of any cellular function
RNA
Protein
Interaction
DNA
others
By their friends, you shall know them!
Transcription factor: DB + AD DB: usually fuse to bait protein AD: prey protein or a cDNA libray
Yeast Two-hybrid
Basis of the Two-Hybrid System. (GIBCO BRL® instruction manual)
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Softwares
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AutoDock Dock GOLD ……
surface plasmon resonance technology (SPR) 表面等离子共振技术
20世纪 年代发展起来的一种基于 世纪90年代发展起来的一种基于 世纪 年代发展起来的一种基于SPR检测生物传 检测生物传 感芯片（ 感芯片（biosensor chip）上配位体与分析物作用的 ） 生物分子检测技术 SPR利用金属膜／ 利用金属膜／ 利用金属膜 液面界面光的全反 射引起的物理光学 现象来分析分子 相互作用
Fluorescence Resonance Energy Transfer (FRET)
Fluorescence
GFP and other FPs (fluorescent proteins)
FRET: principle
• FRET is a distance-dependent physical process by which energy is transferred nonradiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor)
Yeast Two-hybrid
a gold standard for detecting interactions in vivo advantages: in vivo high-throughput screenting disadvantages: yeast (cellular environment; protein modification) overexpressed protein not working with membrane proteins need to be confirmed by other methods
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YFP
His6
CFP
S S Y Y S G
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YFP
His6
Docking
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Give the 3D structure of a protein target, compounds can be designed using bioinformatics methods to fit in a cavity, which is called "docking". Docking is an energy-based operation for exploring the binding modes of two interaction molecules. The treatment ends when a minimum of energy is obtained for the complex.
surface plasmon resonance technology (SPR) 表面等离子共振技术
原理：偏振光在玻片 金膜界面全反射 金膜界面全反射， 原理：偏振光在玻片-金膜界面全反射，激发金膜中 的自由电子产生表面等离子体。 的自由电子产生表面等离子体。当光波与表面等离 子体的振频相等时，二者发生谐振，光能被吸收。 子体的振频相等时，二者发生谐振，光能被吸收。 当入射光波长固定时，反射光强度是入射角的函数。 当入射光波长固定时，反射光强度是入射角的函数。 反射光强度最低时所对应的入射角称为“共振角” 反射光强度最低时所对应的入射角称为“共振角”。 实验中，根据不同情况下共振角的位置变化， 实验中，根据不同情况下共振角的位置变化，来反 映界面上液体的变化。 映界面上液体的变化。 生物分子的相互作用将导致金属薄膜表面介电性质 介电常数、折射率）发生改变。 （介电常数、折射率）发生改变。
Detecting methods
• Monitor changes in donor fluorescence 检测供体 荧光 • Monitor changes in acceptor fluorescence 检测受 体荧光 • Simultaneously measure changes in both donor and acceptor fluorescence using spectral imaging 同时检测受体和供体荧光
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Goals of Docking
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To study ligand-protein, protein-protein and protein-DNA interacitons The influences of mutations on the interaction Drug screening