Molecular biotechnology gives us the tools to study proteins

production of antibodies/antigens
e.g. Hepatitis vaccines

determine protein function

Mutants to map functional

domains (i.e. which parts of a protein are responsible for folding, stability, function etc.)

crystallization protein-protein interactions

Protein-protein interactions make up biological machines that are like intricate three-dimensional jigsaw puzzles
-Wim Van Criekinge and Rudi Beyaert

Protein-protein interactions

An essential part of nearly every cellular process (DNA replication, transcription, splicing, translation) Structural protein interactions required for cell structure and division; signal transduction complexes required to respond to external stimulii May be transient or long-lasting Without these interactions a cell would be deaf and blind and would not survive Understanding the interactions a protein makes can give you information about protein (and therefore gene) function Failures in protein interaction networks are known to contribute to many diseases; understanding them may lead to new treatment methods Much data catalogued through MIPS http://mips.gsf.de/)

Protein-protein interactions make up biological machines that are like intricate three-dimensional jigsaw puzzles (Wim Van Criekinge and Rudi Beyaert) The interlocking protein components assemble and disassemble over time in response to complex signals ***Alteration/disruption of protein-protein interactions is known to contribute to many diseases.

The manipulation of protein-protein interactions that contribute to disease is a potential therapeutic strategy.

http://www.eiu.edu/~eiuchem/faculty/prot einproteininteractions_xp.gif

Extracellular Matrix

-DAG
SG Complex D A G

Na Ch

SP

SG

CYS
Dystrophin (Dp427)

Syn
DB

The Dystrophin Protein Complex

http://www.mshri.on.ca/pawson/images/specificity/fig2.jpg

Please visit the following link and watch the entire video

http://www.dnalc.org/ddnalc/cell_signals/index.html

How do you identify interacting proteins?

Traditionally, protein-protein interactions were identified using Biochemical approaches (protein affinity chromatography, affinity blotting, immunoprecipitation and cross-linking). You can also perform a protein probing experiment using a labeled (radioactive) protein as a probe to screen a Protein expression library One fairly new method (last 10 years) is the Yeast Two Hybrid Assay This assay can identify new interacting proteins and can also characterize known interaction protein couples.

Methods to study protein interactions

Co-immunoprecipitation (CoIP/pulldown) Yeast Two Hybrid Assay (Y2H) Tandem Affinity Purification (TAP)

Background Information: Galactose Metabolism

In yeast, the genes required for galactose metabolism are controlled by 2 regulatory proteins: GAL4 GAL80 When Galactose is present in the media, the GAL4 protein binds to the Upstream Activating Sequence (UAS) of at least 20 known galactose-responsive genes (including the GAL1 gene). UAS sequences are recognized by specific transcriptional activators (i.e. GAL4) that enhance transcription In the absence of galactose, GAL80 binds to GAL4 blocking transcriptional activation.

The GAL4 Protein

The intact Gal4 protein is transcriptional activator with two functionally distinct domains 1. DNA-binding domain, DNA-BD 2. activating domain, AD Normal role of GAL4 protein is to bind the GAL1 Upstream Activating Sequence (UAS) element (with the DNA -BD) and activate transcription (with the AD) from the adjacent promoter.

3
Downstream

GAL1 gene

The Yeast Two Hybrid System

2 Major Concepts: 1. The 2 domains of the GAL4 protein are physically separable. 2. The yeast are engineered so the GAL4 protein activates transcription of reporter genes.

YFP

The GAL4 BD is fused to YOUR FAVOURITE PROTEIN (YFP or bait) This hybrid protein can bind to the GAL1 UAS but cannot activate transcription without the activation domain

Library protein

The GAL4 AD is fused to library proteins (prey). This hybrid protein cannot localize to the UAS by itself and thus does not activate transcription. CAN YOU GUESS WHERE THE NAME YEAST TWO HYBRID ORIGINATED?

The GAL4 BD is incapable of activating transcription unless physically associated with a GAL4 activating domain. BUT, THIS ASSOCIATION DOES NOT HAVE TO BE COVALENT!!!

YFP

Library protein

If YFP and the library protein do not interact, there will be no reporter activation

If YFP interacts with one of library proteins, the GAL4 BD and the GAL4 AD are brought into close proximity GAL4 function is restored!! expression of the reporter genes occurs Expression of HIS reporter will confer prototrophy to his- auxotroph Expression of beta-galactosidase can be assayed using X-GAL or ONPG. http://www.sumanasinc.com/webcontent/animations/content/yeasttwohybrid.html

Yeast Two Hybrid Assay

Overview:

if two proteins interact, then a reporter gene (e.g. -gal) is transcriptionally activated, and you see a coloured product on specific media GAL4 is a transcriptional activator protein involved in galactose metabolism in yeast It consists of a DNA binding domain (BD) and an activation domain (AD) BD interacts with promoter region (UAS); AD interacts with polymerase and initiates transcription

Background:

key points:

BD and AD can be physically separated Yeast are engineered such that the GAL4 protein drives transcription of a reporter gene (gal)

BD fused to YFP (bait); can bind DNA but not activate transcription without AD AD fused to library proteins (prey); will not be localized to correct promoter region without BD and so will not activate transcription alone Bait-prey interaction brings BD and AD close enough together to turn on transcription of reporter gene

Experiment is performed with yeast that: 1. have deletions in the GAL4 and GAL80 genes to avoid interference by endogeneous (produced by yeast) GAL4 and GAL80 2. Have HIS (nutritional) and lacZ (colour) reporter genes downstream from the GAL1 UAS 3. Are His- auxotrophs (i.e. cannot produce histidine on their own) Axotroph: i) An organism with specific nutritional requirements ii) The inability of an organism to synthesize a particular organic compound required for its growth Prototroph: Dont have specific nutritional requirements. To make it simple you could think that a prototroph is the opposite of auxotroph (not exactly the right expression, dont say it like this in the test) When the HIS3 gene is turned on it confers prototrophy to His

http://orders.clontech.com/clontech/techinfo/vectors/catmm.shtml

For the purposes of the Y2H system, the coding sequences for the GAL4-BD and the GAL4-AD are separated and expressed from different plasmids.

Vector

DNA binding Activation domain vector Domain vector Trp -yeast cells containing this vector can survive in Trpmedia Leu -yeast cells containing this vector can survive in Leu- media

Chromosomal reporter genes (in the yeast cells) His3, LacZ -if activated, His3 gene will confer prototrophy on a his3 auxotroph B-Gal is produced (Xgal can be cleaved to give a blue colour)

Selection Marker Purpose

What happens when we transform these plasmids into yeast??

Choose a yeast strain that is trp-, leu- and his- and contains the his and lacZ reporter genes (like CG1945 or Y190)

No vectors

BD vector

AD vector

BD vector AD vector No interaction

BD vector AD vector Interaction

= DEATH

What happens when we transform these plasmids into yeast??

Choose a yeast strain that is trp-, leu- and his- and contains the his and lacZ reporter genes (like CG1945 or Y190)

No trp No leu No his

= DEATH

What happens when we transform these plasmids into yeast??

Choose a yeast strain that is trp-, leu- and his- and contains the his and lacZ reporter genes (like CG1945 or Y190)

No trp No leu No his

BD vector (trp) No leu No his

= DEATH

What happens when we transform these plasmids into yeast??

Choose a yeast strain that is trp-, leu- and his- and contains the his and lacZ reporter genes (like CG1945 or Y190)

No trp No leu No his

BD vector (trp) No leu No his

AD vector (leu) No trp No his

= DEATH

What happens when we transform these plasmids into yeast??

Choose a yeast strain that is trp-, leu- and his- and contains the his and lacZ reporter genes (like CG1945 or Y190)

No trp No leu No his

BD vector (trp) No leu No his

AD vector (leu) No trp No his

BD vector (trp) AD vector (leu) No interaction (no his) No B-gal = DEATH

What happens when we transform these plasmids into yeast??

Choose a yeast strain that is trp-, leu- and his- and contains the his and lacZ reporter genes (like CG1945 or Y190)

No trp No leu No his

BD vector (trp) No leu No his

AD vector (leu) No trp No his

BD vector (trp) AD vector (leu) No interaction (no his) No B-gal

BD vector (trp) AD vector (leu) Interaction (his) B-gal

= DEATH

Where do we start?? Preparing your BAIT

Create your bait construct clone your favourite gene into the GAL4-BD vector (RT-PCR) (Draw it here) It should have: All necessary sequences to be cloned in bacteria antibiotic resistance gene (e.i kan), Ori, MCS) and all necessary sequences to be expressed in yeast (2, promoter upstream of GAL4BD, translation termination/PolyA sequences, nutritional marker (e.i trp) + GAL4BD gene upstream and close to MCS so that you can fuse YFP to it. See generic yeast expression vector (last lecture) and slide 17 (this lecture) Check your bait construct any mutations? (sequence your construct) is the protein stably expressed in yeast? (transform your vector into yeast and test for protein expression by Western analysis)

Western Blot Analysis of Bait Protein

Gal4-dbn (~88kDa)

Antibody: Anti-Gal4 Binding Domain

Auto-activation test
Does your bait construct activate the reporter genes by itself (i.e. without an activation domain)? Transform your bait into yeast alone (without the library) and assay for reporter gene expression (his3 and lacZ). What do you want to see?

Can we use a construct that auto-activates the Y2H system?

Remember this??
YFP

The GAL4 BD is fused to YOUR FAVOURITE PROTEIN (YFP or bait) This hybrid protein can bind to the GAL1 UAS but cannot activate transcription without the activation domain

Positive Control?
Do you have a positive control (i.e. a protein that is known to interact with your bait)? Not every experiment will have a known positive control. If you do, clone the interacting protein into the GAL4-AD vector and transform it into yeast with your bait protein Assay for reporter gene expression If the reporter genes are expressed, your protein is folded properly (this is wonderful to know!!!)

What Library Will You Choose?

Characteristics of the library: (draw an example of a vector here)

Remember: Use a library prepared from a tissue in which your favourite protein is known to be biologically relevant!!

Characteristics of the library: cDNA expression library all proteins are fused to GAL4 activation domain (draw an example of a vector here)

What Library Will You Choose?

The vector should the same kind of sequences as the GAL4BD vector but: Different nutritional marker for yeast (e.i leu) Different antibiotic resistance gene for bacteria (e.i amp) Each protein in the library inserted in the MCS (prey) The GAL4AD instead of the GAL4BD Note: Each clone in the library will have a different prey protein, each will have a different plasmid
All plasmids in the different clones will all have same main sequences except for the prey protein sequence.

Remember: Use a library prepared from a tissue in which your favourite protein is known to be biologically relevant!!

Its Finally Time for the Actual Experiment!!!

Important Note: Yeast, unlike bacteria, can support the propagation of more than one plasmid having the same replication origin Yeast transformation LiAc (Lithium/Acetate) transformation Sequential transformation: 1st Transform the vector containing the bait into your yeast, grow up a liquid culture 2nd Transform the library (the vectors containing all the different prey proteins) into those yeast cells Simultaneously transformation (bait and library at the same time) simpler lower efficiency (lower probability that a particular yeast cell will take up both plasmids)

What media will you use for plating??

Its Finally Time for the Actual Experiment!!!

Important Note: Yeast, unlike bacteria, can support the propagation of more than one plasmid having the same replication origin Yeast transformation LiAc (Lithium/Acetate) transformation Sequential transformation: 1st Transform the vector containing the bait into your yeast, grow up a liquid culture 2nd Transform the library (the vectors containing all the different prey proteins) into those yeast cells Simultaneously transformation (bait and library at the same time) simpler lower efficiency (lower probability that a particular yeast cell will take up both plasmids)

What media will you use for plating?? (trp-, leu-, his-)

Experiment Continued

Plate your transformed cells on trp-, leu-, his- media

Let them grow for up to 10 days (some weak positives will exhibit slow growth)

If the colony represents a positive protein-protein interaction, it should 1) Grow on his- plates 2) Produce B-galactosidase and cleave X-gal to generate a blue colour Pick the isolated his+ colonies

Perform a B-galactosidase test

Place a piece of filter paper on top of an agar plate Streak the yeast clones onto the filter paper

Let the yeast grow into a thick inoculation line on the filter paper
Remove the filter paper from the plate Drop the filter paper into liquid nitrogen to break open the yeast cells (this will release the B-galactosidase from the cells) Place the filter in a Petri dish with a buffer solution containing X-gal Watch to see which clones cleave X-gal to turn blue (this may take a few hours!) The clones that turn blue indicate that the LacZ reporter gene was activated (producing B-galactosidase) due to a positive protein-protein interaction

What Next??
BD-YFP vector AD-X vector Interaction BD-YFP vector AD-Y vector Interaction

What we have: Many yeast clones!!!

What we want: To determine the nature of the interacting protein

AD-X vector

AD-Y vector

But yeast are not as nice to work with as bacteria! So lets make our job easier!!!

Separating the Two Hybrid Vectors

BD-YFP vector AD-X vector Interaction

Yeast cells

Make DNA (i.e. yeast plasmid minipreps will contain both plasmids)

Transform the DNA into E. coli

Separating the Two Hybrid Vectors

BD-YFP vector AD-X vector Interaction

Yeast cells

Make DNA (i.e. yeast plasmid minipreps will contain both plasmids)

Remember, bacteria can only support one plasmid

Transform the DNA into E. coli 50% BD-YFP AD-X 50%

Plate onto

Separating the Two Hybrid Vectors

BD-YFP vector AD-X vector Interaction

Yeast cells

Make DNA (i.e. yeast plasmid minipreps will contain both plasmids)

Remember, bacteria can only support one plasmid

Transform the DNA into E. coli 50% BD-YFP AD-X 50%

Plate onto LB/Amp Cells with BD-YFP cannot grow Cells with AD-X can grow

Final Steps
Miniprep all of the AD plasmids from all of your bacterial clones Sequence each of the plasmids -use a primer in the AD, this will give you sequence from your unknown genes!!!! Confirm the interaction using independent techniques!!!

Disadvantages to the Y2H System

fusion to the GAL4 Domain might change the actual conformation of the bait and/or prey, -could interfere with protein binding sites -could alter function YFP must be able to fold correctly and exist as a stable protein inside the yeast cells. Posttranslational modifications (eg. disulfide bridges, glycosylation, phosphorylation) do not occur in yeast. Any interactions that depend upon these modifications will not be detected. Some proteins might become toxic when expressed in yeast (i.e. yeast will die).

Disadvantages Continued
Since all combinations of protein-protein interactions are assayed, there is a very good possibility of identifying false positives. Explanation In the cell, time and space constraints govern potential interactions i.e. it is possible that 2 proteins shown to interact by Y2H are: not expressed at the same time in the cell not expressed in the same cellular compartment or cell type

Once two interacting partners are identified, the biological relevance of the interaction needs to be determined.

Advantages to the Y2H system

Can find new interacting protein partners Allows for the analysis of known protein-protein interactions in vivo technique using the yeast host cell as a live test tube Weak and/or transient can be readily detected (thanks to reporter genes!) When an interacting protein is identified, the corresponding gene is cloned at the same time!!!!!!!

ALWAYS REMEMBER: any protein-protein interaction discovered using the Y2H method still needs to be validated by other techniques

Co-Immunoprecipitation

Immunoprecipitation is the precipitation of an antigen out of solution using an antibody (recall: affinity purification)

Co-immunoprecipitation identifies interacting proteins that precipitate with your target protein
Figures from: www.soi.wide.ad.jp

Affinity Chromatography Methods

Co-IP Video
Watch this video about Co-IP from minute 1:OO. Please disregard what is explained before minute 1:00, we are not studying the TNT system.
VIDEO
http://www.promega.com/resources/multimedia/protein-expression-purification-and-analysis/coimmunoprecipitation-to-study-protein-proteininteractions-using-the-tnt-systems/

Note that the antibody used for immunoprecipitation is directed to protein 1 (protein of interest) and the the antibody used for Western Blot is directed to protein 2 (suspected interacting protein). Why?

Co-IP flowchart

Cell contains two interacting proteins Lyse Add Ab directed at protein of interest Add Ab binding beads IP protein of interest Wash and collect beads Western blot analysis of IPd proteins; probe with Ab vs suspected interacting protein This is considered the gold standard of protein-protein interaction studies, especially if done with endogenous (i.e. not tagged, not overexpressed) proteins

Tandem Affinity Purification (TAP-tagging)

Like Y2H, involves making YFP into a fusion protein; fusion is to affinity tags rather than transcriptional activator protein Tendem = 2; YFP is fused to a double tag that allows two purification steps in one experiment tag consists of three components: a calmodulin-binding peptide, a tobacco etch virus (TEV) protease cleavage site and Protein A as an immunoglobulin G (IgG)binding domain.

Tandem Affinity Purification

Cells or organisms are generated that contain TAP-tagged protein(s). Extracts are then prepared and run on the first column: IgG beads. TEV protease cleaves the immobilized multi protien complexes. Second round of binding is carried out on a calmodulin bead column. Native complex is eluted by chelating calcium using EGTA. Complex analyzed by SDS-PAGE or mass-spectrometry

TAP tagging: pros and cons

Advantages Can determine protein interactions quantitatively and In Vivo; proteins are generally expressed at regular cellular levels Prior knowledge of complex composition is not required Disadvantages Addition of tag could interfere with binding of protein to interacting partners Tag may affect protein expression levels May not be able to detect transient interactions (2 rounds of washing); higher specificity examination of permanent interactions

Review: Protein-Protein interactions

Understanding protein-protein interactions is essential to understanding cell function Recombinant DNA technology gives us new ways to study protein-protein interactions 3 methods discussed (with pros & cons of each)

Co-IP Y2H TAP-tag

Mass Spectrometry (MS)

premise: proteins are identified by the overlap between a set of identified and predicted signature peptides basic protocol: whole proteins broken down to peptide fragments by trypsin digestion peptides separated by mass to charge (m:z) ratio measured as peaks on chromatogram

Trypsin cuts proteins specifically on the C-terminal side of every lysine and argine in the amino acid sequence (except when these bonds are to prolines).

several peaks from each protein required to identify the protein

Parts of a Mass Spectrometer

Ionization device m from solid phase (2D gel or affinity chromatography column) into gaseous ion phase Examples: o matrix-assisted laser desorption ionization (MALDI) o electrospray ionization (ESI) Separation Chamber ions move through vacuum according to charge & mass separated based on time of flight (TOF) Detector can separate peaks for >10,000 different molecular species

Parts of a Mass Spectrometer

Ionization Device: Sample goes from solid phase to gaseous phase. Can be directly connected to a chromatography column Separation Chamber: Peptides are separated by mass/size ratio here. 1st-ions are accelerated so they have the same kinetic energy. 2nd- ions are deflected by a magnetic field according to their masses (the lighter the more deflected) and the amount of + charges on the ions (the more + ions the more deflected). Vacuum is important.

www.chemguide.com

Detection: T.O.F : The to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds).

Data Analysis

protein identities are computed from peptide m/z spectra by comparison with a database of predicted spectra in the proteome databases assembled by in silico trypsin digestion of theoretically translated ESTs(Expressed Sequence Tags) or cDNAs

Data Analysis
Mimicking the experiment in silico

A theoretical mass spectrum is constructed (using same enzyme used for digestion of real sample) and compared with the measured mass spectrum. The entries in the protein sequence collection are ranked according to how well they match the experimental data

Higher-tech MS tandem mass spectrometry

(MS/MS) collision chamber between ion source and detector ions collide with nitrogen or argon gas at low pressure; causes fragmentation of peptide backbone subfragments are separated by TOF and detected peaks are separated by characteristic widths that correspond to the aa that is removed from either end of the peptide in the collision chamber

Liquid chromatography mass spectrometry (LC/MS/MS)

for characterization of entire proteome at once

eliminates need for prior electrophoretic/

chromatographic separation Combines the s of LC with the mass analysis capabilities of MS Samples of complex biological fluids like human serum may result in over 1000 proteins being identified (Sample needs to be separated in an SDS-PAGE first)

Protein Interaction Databases

http://mips.gsf.de/proj/ppi/

Additional References (not required reading): http://www.ploscompbiol.org/article/info:doi/10.1371/ journal.pcbi.0030042 Deciphering ProteinProtein Interactions. Part I. Experimental Techniques and Databases

http://www.ploscompbiol.org/article/info:doi/10.1371/ journal.pcbi.0030043
Deciphering ProteinProtein Interactions. Part II. Computational Methods to Predict Protein and Domain Interaction Partners