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