INTERACTIVE PROTEOMICS – TECHNIQUES FOR EXPLORING THE SOCIAL NETWORK OF CELLS

Karobi Karobi Moitra (Ph.D)Moitra (Ph.D)NCI Frederick , NIHNCI Frederick , NIHCancerCancer Inflammation ProgramInflammation ProgramHuman Genetics SectionHuman Genetics SectionFrederick MD.Frederick MD.

Proteome: the entire protein complement of a cell , tissue, or organism

The proteome is DYNAMIC !

Why is the proteome dynamic ?

Proteins can be:

Synthesized

Modified by post-translational modifications

Undergo translocations within the cell

Degraded

Examination of the proteome of a cell is liketaking a “snapshot” of the protein environment

at any given time

FREEZE FRAME………

Proteomics: is the large scale characterization of thisproteome

Proteome: the entire protein complement of a cell , tissue, or organism

Why do we need to characterize the proteome?

• To obtain a more global and integrated view of biology bystudying all the proteins of a cell rather than each oneindividually

• To create a complete three-dimensional (3-D) map of the cellindicating where proteins are located

The Google Earth Analogy

Global ProteinLandscapes

Different areas of study are now grouped underthe rubric of proteomics include:

Protein modifications

Protein function

Protein localization

Protein-protein interactions (Interactive proteomics)

THE ABILITY OF A PROTEIN TO BIND OR INTERACT WITH ANOTHER PROTEINOR PROTEINS

WHAT IS INTERACTIVE PROTEOMICS OR PROTEIN – PROTEIN INTERACTIONS?

Types of protein interactions :

Permanent interactions

Transient interactions

From the need for more -omics came the terminteractome

THE INTERACTOMECOMPLETE PROTEIN INTERACTION NETWORK OF A CELL ORAN ORGANISM

Just as humans don’t thrive when isolated from other humans - the same can be

said for proteins !

WHY ???

Proteins interact with other proteins to provide :

Structural integrity to the cell (e.g., actin filaments)

Transport molecules (e.g.,Transporters)

Propagate signals (e.g., kinases)

Transcribe DNA, translate other proteins etc.

….there is no protein discovered yet that acts on its ownwithout interacting with any other entity !

The proteome is DYNAMIC

Proteins can interact or bind with other protein(s)

This ‘social’ network is called the INTERACTOME

The entire protein complement of a cell, tissue, or organism is calledthe PROTEOME

….in the real world you have to interact with people to learn the ‘social dynamic’

…. in the protein world you would have to know how proteins(and other components of a cell) interact with each other in order to explore

the ‘cellular’ dynamic

You have been asked to find the interacting partners ofa protein named ‘C3PO’. Your first task is to find outeverything you need to know about this protein in order toundertake this study.

Your tool is the internet, which sites you would go to andwhat information might you obtain from these sites to getthe relevant background knowledge you would need tocarry out the study?

Partial List of potential websites:

www.google.com

www.ncbi.nlm.nih.gov/

http://www.ensembl.org/index.html

www.expasy.ch

http://www.expasy.org/links.html

And a lot more links from this page………

1.Clone and express the protein (C3PO) in an expression system of your choice

2. Optimize protein expression

3. Decide which techniques you would use to study protein-protein interactions

TECHNIQUES USED TO STUDYPROTEIN-PROTEIN INTERACTIONS

A. Standard techniques to probe protein-protein interactionsAffinity purificationMass SpectrometryTwo-Hybrid AssayPhage Display

B. In Vivo ImagingFluorescence Microscopy

C. Biophysical ApproachesProtein Co-crystallization

D. MicroarraysHigh Density Protein Microarray

E. Computational/Bioinformatics MethodsComputer programs that simulate protein-protein interactionsPrediction of co-evolved protein pairs based on similarphylogenetic trees

Affinity purificationBasic Principle:Historically affinity purificationwas based on a specific biologicalinteraction such asenzyme-substrate.In a broader sense it may meanChemical/biological affinity.

Stationary/solid phaseDynamic/liquid phase

Immunoprecipitation (IP) is the technique of precipitating a protein antigenout of solution using an antibody that specifically binds to that particularprotein.

Immunoprecipitation

A. Standard techniques to probe protein-protein interactions

Immunoprecipitation/coimmunoprecipitation

Basic Principle:

YX A

B

Run gelVisualize proteinExcise bandDigestMS

YX

BA

lysate

YX

Post- ip

YX A

BCell lysis

Freeze thawLysis bufferHypotonicMild detergent(Ripa, NP40)

YX

A

ProteinA/G beads(binds to Fc of Ab)

Aelute wash

Low pH(change pH)SDS loading buffer

Lysis buffer

Disadvantage: An antibody to the specific protein of interest is required

Solution: We can tag our protein of interest with an epitope tag

(i) Single Tag FLAG tag , c-Myc tag, GST tag, His tag etc.

(ii) Tandem affinity purification TAP tag

Epitope Tagging :Antibody recognizes a specific portion of the protein - epitope.

Target protein Flag tag

Anti-Flag Abcoupled tobeads

Associated proteins

(i) Single Tag

Attaching the Tag :

Transfect into cells to express the protein

Clone into vector

Note:Tags can be Nterminal or C terminaldepending on where thefunctional region of the protein is located. Taggingclose to the functional regionmay interfere with binding sites.

Histidine Tag

Nickel column

OrImidazole

Imidazolegroups

Imidazole groupscan form a coordinatecovalent bond with metals

Controls :

Transfection:Untransfected cellsVector transfectedKnown positive control

Pulldown:Vector transfected cellsKnown positive control

Transfect cells48-72hrs

(for peptide purification- antibody productionAlso complex pulldown)

(Complex pulldown)

(Low conc. to wash out non-specific binding)

OrMechanical lysisFreeze-thaw method

(Compete off His taggedProtein)

His-tagged proteinbinds to Ni column

(ii) Tandem Affinity Purification :

2 step purification :

1. Purify through Protein A tag on a IgG-sephrose column

2. Purify through Calmodulin binding domainon a Calmodulin-sepharose column

2 step purification removes a lot of the background / non-specific protein binding

Tandem affinity purification (TAP)

TAP TAG

(tobacco etch virus)

IgG-sepharose bead

Calmodulin-sepharose beads

Elute

Target protein

IgG-sepharose bead

IgG-sepharose bead

TEV cleavage Step 1 Purify protein by passing through IgG column and elute with TEV

Step 2Purify protein by passingThrough Ca + calmodulin columnAnd elute with EDTA

(EGTA)

The TAP strategy

Evaluation of a Co-IP Captured interaction

1.Confirm that the co-precipitated protein is obtained only by the antibody against the target , try and use monoclonal antibodies , if using polyclonals purify the antibody using an affinity column containing pure target

2. Use an antibody against the co-precipitated protein to co-IP the same complex

3. Determine that the interaction takes place in the cell and not as a consequence of cell lysis, use co-localizatiion or mutation studies to confirm interaction.

4. Run a negative control IP with unrelated antibodies.

(i) Single Tag (ii) Tap tag (iii)Photochemical/chemical crosslinking

Photochemical / Chemical Crosslinking of Proteins

The interactions or proximity of proteins can be studied by the clever useof crosslinking agents. Protein A and B may be quite close to each other in a cell and a chemical crosslinker can be used to probe the protein-protein interaction by linking them together, disrupting the cell and detecting thecrosslinked proteins.

A B

Diazirine based photo crosslinking

Cells grown with photoreactive diazirine compounds

Diazirine incorporated into protein

UV light

Diazirines activated and bind to interacting proteins (within a few angstroms)

A B

Chemical Crosslinking

• Covalently links distinct chemical functional groups & can detect both stable and transient interactions

• If 2 proteins physically interact with each other they can be covalently crosslinked

Crude cellular extract + crosslinking agent (maleimides -SH reactive groups would form disulphide bonds between proteins )

IP

Recover complexes

Cleave with DTT, BME which would break disulphide bonds

A B

Example : SMCC, succinimidal trans -4 (maleimidemethyl) cyclohexane-1- carboxylate

(a) Epitope tagging

(b) Chemical cross-linking

(c) TAP tag approach.

You have your putative protein -complex of interest

how would you identify the individual proteins that make up

this complex ?

SCHEMATIC DIAGRAM OF PROTEIN IDENTIFICATION

(can ID only 50-60 aa)

MASS SPECTROMETERY

Protein structural information : peptide mass amino acid sequences

Type and location of post-translational modifications

Experimental Design

Immuno-precipitation

Examine complexes by SDS-PAGE

Mass Spectrometry

In-gel digestionwith trypsin (K/R)

Extract peptides

MALDI-TOF/TOF

LC- TANDEM MS

PROTEIN IDENTIFICATION BY PEPTIDE MAPPING(MALDI-TOF)

MALDI-TOF Matrix assisted laser desorption ionisation- time of flight

Soft ionisation technique suitable for fragile biomolecules like peptides

Basic Principle of Mass Spectrometry

How it works :

The amount of deflection for asideways force depends on theMass of the ball (acceleration constant)

Acceleration - knownForce - knownMass - can be calculatedForce= mass x acceleration

Peptides + matrix (matrix protects peptides from the direct laser beam and help absorption of laser energy)

Spotted onto a target plate

Ionised by laser beam (charge needed for deflection by electric field)

Ionised particles enter flight tube

Charged peptides move to other side of tube according to mass

Peptides hit the detector and time of flight (TOF) is recorded (to calculate speed)

Opposite charge

(known force)

(speed)

The computer generates a mass spectrum, with each peak representing the mass to charge ratio (m/z) as a function of the % relative intensity (abundance)of the detected peptide

The list of experimental peptide masses is compared against the theoreticaltryptic digest of every protein in a protein database.

When the experimental data matches the theoretical, the protein is identified.A probability based scoring system is used for the search, indicating the ‘hit’ is not a random event.

PROTEIN IDENTIFICATION BY TANDEM MASS SPECTOMETRY (MS/MS)

If the protein cannot be identifed via the peptide mass profile (eg the protein may not be listed in any database) then Tandem (MS/MS) maybe used to obtain an amino acid sequence.

Q1- 1st mass analyser (quadrupole) isolates peptide ion of interest

Q2- Collision chamber peptide ion collides with neutral gas molecules (helium,nitrogen or argon) and fragments into smaller pieces

Q3- 2nd analyser (TOF) leads to detector which gives a product profile (aa sequence)

Fragments the peptides into the smallest length to ID short sequences

A. Standard techniques to probe protein-protein interactionsAffinity purification Mass Spectrometry Two-Hybrid AssayPhage Display

B. In Vivo ImagingFluorescence Microscopy

C. Biophysical ApproachesProtein Co-crystallization

D. MicroarraysHigh Density Protein Microarray

E. Computational/Bioinformatics MethodsComputer programs that simulate protein-protein interactionsPrediction of co-evolved protein pairs based on similarphylogenetic trees

• Test the association of two specific proteins that are believed to interact on the basis of other criteria.• Define domains or amino acids that are critical for the

interactions of two proteins that are known to interact• Screen libraries for proteins that interact with a

specific protein.

Yeast 2-hybrid assay

Basic Principle of 2- Hybrid Assays

The basic premise of a 2- hybrid assayis that a prey protein is detected withthe help of a bait protein.

A transcription factor is split into 2parts a DNA binding domain - BD andan activation domain AD.

The BD is engineered to bind to thebait and the AD is engineered tobind to the prey.

Only if the bait and the prey proteininteract will the transcription factorcome together and transcribe a reportergene.

Activating domain

Bindingdomain

Probing Protein-Protein Interactionswith the Yeast 2-Hybrid Assay

Activation Domain Protein YDNA Binding Domain Protein X +

YES NOHIS3 HIS+ his-

lacZ Blue white

ADE2 Whit red

Split-Ubiquitin Membrane Yeast Two-Hybrid System

Drawbacks of typical Y2H necessitated the split-ubiquitin Y2H

1.Hybrid proteins are directed towards the nucleus so proteins that fold incorrectly in nucleus are excluded from the method (integral membrane proteins).

2. Interactions dependent on post-translational modifications ( in ER) won’t take place.

3. Interactions mediated by the amino-terminus may not work because the transcription factor domain blocks accessibility.

Split-Ubiquitin Membrane Yeast Two-Hybrid System

1. Contains 2 fragments of ubiquitin broughttogether upon interaction of the

2 proteins.

2. The bait protein X is fused to the C-term ofubiquitin (Cub) followed by a TF

3. The prey protein Y is fused to N-term ofubiquitin (NubG)

4. The 2 plasmids are introduced into yeastL40 strain.

5. Interaction of X and Y leads to theassembly of ubiquitin and the proteolytic

release of transcription factor (by ubiquitinproteases).

6. The transcription factor activates the 2reporter genes lacZ and His3 so

the interactions can be monitored bygrowing yeast in histidine deficient

media or by performing an X-gal test forthe expression of beta galactosidase.

XY

BaitPrey

Transcription factor

A. Standard techniques to probe protein-protein interactionsAffinity purification Mass Spectrometry Two-Hybrid Assay Phage Display

B. In Vivo ImagingFluorescence Microscopy

C. Biophysical ApproachesProtein Co-crystallization

D. MicroarraysHigh Density Protein Microarray

E. Computational/Bioinformatics MethodsComputer programs that simulate protein-protein interactionsPrediction of co-evolved protein pairs based on similarphylogenetic trees

PHAGE DISPLAY

In phage display new genetic material is inserted into a phage gene andthe bacteria process the new gene so that a protein/peptide is made and exposed on the phage surface (due to a tag which only expresses on the cell surface).

A population of bacteriophages display hundreds/millions of protein -one protein per phage.This is called a phage display library.

Basic Principle:

This library can be exposed to an immobilized target protein and some members willbind to the target. The immobilized target is then washed to remove non/loose bindingphages. The DNA of phages that bind can be sequenced to identify the gene/protein.

B. IN VIVO IMAGING

Fluorescence Microscopy

Basic Principle

Fluorescent molecules are irradiatedwith high intensity light.

When these molecules absorb a photon of light an electron is boosted up to a higherenergy orbit creating an excited state

When this electron returns to the ground statea photon of light may be emitted- this iscalled fluorescence.

Fluorophores have distinct excitation andemission spectra.

How can we use fluorescence microscopy to study protein-protein interactions?

1. FRET (Fluorescent Resonance Energy Transfer)

2. BRET (Bioluminescence Resonance Energy Transfer)

1. FRET (Fluorescent Resonance Energy Transfer)

Normally an excited photon returns to the ground state when a photon is emitted.FRET results in the excitation of a nearby acceptor fluorophore which will emit a photon when it goes back to the ground state.

The occurrence of FRET thus results in decreased donor emission and increasedacceptor emission.

FRET is extremely sensitive to the distance among fluorophoresFor CFP and YFP the half maximum distance or Forster radius is 49-52 angstroms

Distance is everything !

Basic Principle of FRET

One probable interaction partner is tagged with CFP the other with YFP.If the 2 proteins interact emission will be observed at 530nm instead of 475nm

CFP YFP

475

Problems of FRET

1. Tissues and cells may be damaged by excitation light

2. Some tissues like the retina and most plant tissues are photoresponsive

3. Photobleaching, autofluorescence or diect excitation of the acceptor fluorophore may occur.

2. BRET (Bioluminescence Resonance Energy Transfer)

In BRET the excitation light is replaced by bioluminescent light fromRenilla luciferase (RLUC)The luciferase is activated by its substrate coelenterazine.

Bioluminescent light

C. Biophysical Approach

1. Protein Co-crystallization

Protein Co-crystallization

-grow crystal

-collect diffraction data

-calculate electron density

-trace chain & generate structure

SNL1 & YPD1 co-crystals

SNL1 and YPD1 are part of a phosphorelaysignal transduction pathway in yeast.these protein can be co-crystalized by usinga phosphate analog (BeF3) which bindcovalently and activates respose regulatorproteins.

(Chooback . L 2003)

YPD - YellowSLN1- Cyan

(Xu et. al 2003)

Co-Crystal structure of YPD1 and SLN1

D. Microarrays

High Density Protein Microarray

Microspots of the captured moleculesare immobilized in rows and columnson a solid support

They are exposed to samples containingthe corresponding binding molecules.

Proteins interact

Readout systems based on fluorescence, chemiluminescence,massspectometry, radioactivity etc. can be used to detectcomplex formation

E. Computational/Bioinformatics Methods

1.Computer programs that simulate protein-protein interactions ie Docking programs like Autodock.

2. Prediction of co-evolved protein pairs based on similar phylogenetic trees

This method involves using a sequence search tool such as BLAST for findinghomologues of a pair of proteins, then building multiple sequence alignments withalignment tools such as Clustal. From these multiple sequence alignments,phylogenetic distance matrices are calculated for each protein in the hypothesizedinteracting pair. If the matrices are sufficiently similar they are deemed likely tointeract.

A. Standard techniques to probe protein-protein interactionsAffinity purification Mass Spectrometry Two-Hybrid Assay Phage Display

B. In Vivo ImagingFluorescence Microscopy

C. Biophysical ApproachesProtein Co-crystallization

D. MicroarraysHigh Density Protein Microarray

E. Computational/Bioinformatics MethodsComputer programs that simulate protein-protein interactions Prediction of co-evolved protein pairs based on similar phylogenetic trees

Humans do not thrive when isolated from others - the same can besaid for proteins !

TO MARGUERITE by: Matthew Arnold (1822-1888)

‘Yes in the sea of life enisled, With echoing straits between us thrown. Dotting the shoreless watery wild, We mortal millions live alone. The islands feel the enclasping flow, And then their endless bounds they know…’

Think-Pair- Share Activity

In light of what you have learnt about proteins today do you thinkthat a protein can function on its own isolated from other proteins?

For or Against

‘O then a longing like despairIs to their farthest caverns sent!For surely once, they feel, we wereParts of a single continent.Now round us spreads the watery plain--O might our marges meet again!’