p-p interaction (ion)
TRANSCRIPT
Determination of Protein-Protein Interaction
周金秋Institute of Biochemistry and Cell Biology
Chinese Academy of Science
Fall 2004
Protein-protein interactions are intrinsic to virtually every cellular process
Stable interaction protein complex
Transient interactionenzyme-substrate
Biological significance
Strength of Protein-Protein Interactions
- Affinity
Kd=[A][B]/[AB], Kd: dissociation constant
Interaction
Streptavidin-biotin bindingAntibody-antigen interaction (good)Antibody-antigen interaction (weak)Enzyme-substrate
Kd
10-14 M10-8 -10-10 M10-6 M10-6 -10-10 M
How to Detect Protein-Protein Interactions(identification/characterzation/manipulation)
• Biochemical approaches
• Surface plasmon resonance
• Genetic approaches
• Two-hybrid
• Traditional co-purification (chromatography on different
matrix, co-sedimentation)
• Affinity chromatographyGST pull down Epitope-tag
• Immunoprecipitation
• Far-Western
Biochemical Approaches
Zuo S, Gibbs E, Kelman Z, Wang TS, O'Donnell M, MacNeill SA, Hurwitz J. (1997) DNA polymerase delta isolated from Schizosaccharomyces pombe contains five subunits. Proc Natl Acad Sci U S A 94:11244-9
• Traditional co-purification (chromatography on different
matrix, co-sedimentation)
• Affinity chromatography GST pull down Epitope-tag
• Immunoprecipitation
• Far-Western
Biochemical Approaches
GST Fusion Protein Purification
binding wash elution
GST Pulldown
binding wash elution
GST Pulldown
binding elution
GST Pulldown
GST Pulldown
binding wash elution
Qi and Zakian (2000) The Saccharomyces telomere-bindingprotein Cdc13p interacts with both the catalytic subunit of DNA polymerase and the telomerase-associated Est1 protein GENES & DEVELOPMENT 14:1777–1788
• Traditional co-purification (chromatography on different
matrix, co-sedimentation)
• Affinity chromatographyGST pull downEpitope-tag
• Immunoprecipitation
• Far-Western
Biochemical Approaches
Epitope Tagging
binding wash elution
Shou W et al (1999) Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 1999 Apr 16;97(2):233-44.
• Traditional co-purification (chromatography on different
matrix, co-sedimentation)
• Affinity chromatography (Epitope-tag) GST pull down
• Immunoprecipitation
• Far-Western
Biochemical Approaches
Immunoprecipitation
binding wash elution
YY
Y Y Y
Co-immunoprecipitation
binding wash elution
YY
Y Y Y
Shou W et al (1999) Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 1999 Apr 16;97(2):233-44.
• Traditional co-purification (chromatography on different
matrix, co-sedimentation)
• Affinity chromatography (Epitope-tag) GST pull down
• Immunoprecipitation
• Far-Western
Biochemical Approaches
1. Proteins are first transferred from gel to membrane.
2. Probed with a pure protein known to interact or putatively interact with one or more proteins transferred to the membrane. The protein probe or "bait" protein is isotopically labeled and the interaction with the "prey" protein is detected on the membrane.
3. Visualize the interaction
Far-Western
A B C
Labeled protein “D”
A B CA B C A B C
• Require antibodies or protein labeling
• Harsh condition
• Provide static “snap-shots” of a dynamic process
• No kinetic parameters of the interactions
Drawbacks
How to Detect Protein-Protein Interactions(identification/characterzation/manipulation)
• Biochemical approaches
• Surface plasmon resonance
• Genetic approaches
• Two-hybrid
SPR is used to monitor interactions occurring in a biospecific surface on a metal layer by measuring changes in the solute concentration at this surface as a result of the interactions.
Surface Plasmon Resonance
Surface Plasmon Resonance
Advantadge of BIAcore SPR
Label-free detection
Biacore does not require any labels or reporter groups to detect biomolecular interactions. Biacore follows every step in a multi-step analysis procedure, in contrast to label-based methods that often only report the final step.
Real time measurement
The progress of interactions is displayed directly on the computer screen in Biacore, as a plot of response (which is directly related to concentration changes at the surface) against time. Immediate feedback on the status of an interaction speeds up assay development and analysis. The results can be processed further after the run, for example to extract kinetic constants for the interaction.
How to Detect Protein-Protein Interactions(identification/characterzation/manipulation)
• Biochemical approaches
• Surface plasmon resonance
• Genetic approaches
• Two-hybrid
Genetic Approach
A
B
C
D
B’
Phenotype
Wild
ty
pe
rad
50
mre
11
Nugent CI et al (1998) Telomere maintenance is dependent on activities required for end repair of double-strand breaks. Curr Biol 1998 May 21;8(11):657-60
Chen et al. (2001) Promotion of Dnl4-Catalyzed DNA End-Joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 Complexes Molecular Cell, Vol. 8, 1105–1115
Genetic Approach
A
B
C
D
B’
Phenotype
Ritchie KB, Petes TD. The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast. Genetics 2000 May;155(1):475-9
Genetic Approach
A
B
C
D
B’
Phenotype
Tel1p
Rad50p/Mre11/Xrs2
How to Detect Protein-Protein Interactions(identification/characterzation/manipulation)
• Biochemical approaches
• Surface plasmon resonance
• Genetic approaches
• Two-hybrid
Two-Hybrid System (Interaction Trap)
• Identification of interacting partners
• Characterization of known interaction couples
• Manipulate protein-protein interactions
lacZUAS
ADDB
lacZUAS
lacZUAS
DB AD
AD
Y
DB
X
lacZ
lacZ
BD
X
Mar
ker1
AD
Y
Mar
ker2
AD
Y
lacZUAS
DB
X AD
Y
lacZ
DB
XDB
X
DB
XA
D
Y
AD
Y
BD
X
Mar
ker1
AD
Y
Mar
ker2
Advantages of Two-Hybrid System
• An in vivo technique using the yeast host cell as a live test tube
• Only the cDNA (full-length or even partial) is needed
• Readily detected weak and transient interactions
• Allow for the analysis of known interactions
• Functional screening of the corresponding gene
AD
Y
lacZUAS
DB
X AD
Y
lacZ
DB
X BD
X
Mar
ker1
AD
Y
Mar
ker2
lacZUAS
DB X
lacZ
DB
X
Auto activation
BD
X
Mar
ker1
AD
Y
lacZUAS
ADY
lacZ
Auto activation
AD
YM
arke
r2
•All proteins are artificially made fusion proteins (chimeras might change the actual conformation of the bait and/or prey)
•Posttranslational modifications do not, or inappropriately, occur in yeast.
•Two-hybrid system needs the fusion proteins to be targeted to the yeast nucleus.
•Auto-activation (switching or "swapping" might provide an empirical way to escape the problem)
•When screening libraries, a good representation is crucial. Many isolates may not represent full-length cDNA.
•The possibility that a third protein Z is bridging the two interacting partners can not be excluded.
•Some proteins might become toxic upon expression in yeast.
•False positive could be due to the so-called time/space constraints
Disadvantages and Drawbacks of Two-Hybrid System
Summary
Biochemicalapproaches
Genetic approachesTwo-hybrid
Thank You
Mr. Chen Yongbin
Reference: Sambrook and Russell, Molecular Cloning A Laboratory Manual, Third Edition (Chapter 18), Cold Spring Harbor Laboratory Press