P-p Interaction (ion)

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

Biological significance

Transient interaction enzyme-substrate

Strength of Protein-Protein Interactions - Affinity Kd=[A][B]/[AB], Kd: dissociation constant Interaction

Kd

Streptavidin-biotin binding Antibody-antigen interaction (good) Antibody-antigen interaction (weak) Enzyme-substrate

10-14 M 10-8 -10-10 M 10-6 M 10-6 -10-10 M

How to Detect Protein-Protein Interactions (identification/characterzation/manipulation)

• Biochemical approaches • Surface plasmon resonance • Genetic approaches • Two-hybrid

Biochemical Approaches • Traditional co-purification (chromatography on different matrix, co-sedimentation) • Affinity chromatography GST pull down Epitope-tag • Immunoprecipitation • Far-Western

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

Biochemical Approaches • Traditional co-purification (chromatography on different matrix, co-sedimentation) • Affinity chromatography GST pull down Epitope-tag • Immunoprecipitation • Far-Western

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-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase and the telomerase-associated Est1 protein GENES & DEVELOPMENT 14:1777–1788

Biochemical Approaches • Traditional co-purification (chromatography on different matrix, co-sedimentation) • Affinity chromatography GST pull down Epitope-tag • Immunoprecipitation • Far-Western

Epitope Tagging

binding

wash

elution

Shou W et al (1999) Exit from mitosis is triggered by Tem1dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 1999 Apr 16;97(2):233-44.

Biochemical Approaches • Traditional co-purification (chromatography on different matrix, co-sedimentation) • Affinity chromatography (Epitope-tag) GST pull down • Immunoprecipitation • Far-Western

Immunoprecipitation

Y

Y

binding

Y

Y

wash

Y

elution

Co-immunoprecipitation

Y

Y

Y

binding

Y

wash

Y

elution

Shou W et al (1999) Exit from mitosis is triggered by Tem1dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 1999 Apr 16;97(2):233-44.

Biochemical Approaches • Traditional co-purification (chromatography on different matrix, co-sedimentation) • Affinity chromatography (Epitope-tag) GST pull down • Immunoprecipitation • Far-Western

Far-Western 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

Labeled protein “D”

A

B

C

A

B

C

A

B

C

A

B

C

Drawbacks • Require antibodies or protein labeling • Harsh condition • Provide static “snap-shots” of a dynamic process • No kinetic parameters of the interactions

How to Detect Protein-Protein Interactions (identification/characterzation/manipulation)

• Biochemical approaches • Surface plasmon resonance • Genetic approaches • Two-hybrid

Surface Plasmon Resonance 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

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’

B C D

Phenotype

mre11∆

rad50∆

Wild type

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’

B C D

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

Tel1p

B’

B

Rad50p/Mre11/Xrs2

C D

Phenotype

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

AD

DB UAS

lacZ

AD

DB UAS

lacZ

XY

UAS

AD

DB

lacZ

Ma rk e r1

BD

X

Ma

rke

r2

AD

Y

AD Y

AD

lacZ rke

r2

UAS

AD

Ma

DB

AD

X DB

Y

AD

XY

Y

DB

rke

DB

Y

BD

Ma

X

r1

X

X

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

Y

AD

DB

AD

XY

lacZ AD

DB

rke

Y

BD

Ma

X

r1

Ma

rke

r2

UAS

X

DB X

UAS

lacZ

DB

BD

Ma

X

rke

r1

Auto activation

X

AD Y

lacZ

AD

Ma

rke

r2

UAS

Auto activation

Y

AD

Y

Disadvantages and Drawbacks of Two-Hybrid System •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

Summary Biochemical approaches

Two-hybrid

Genetic approaches

Thank You Mr. Chen Yongbin

Reference: Sambrook and Russell, Molecular Cloning A Laboratory Manual, Third Edition (Chapter 18), Cold Spring Harbor Laboratory Press