CZ5211 Topics in Computational BiologyCZ5211 Topics in Computational Biology

Lecture 6: Biological Pathways I: Lecture 6: Biological Pathways I: Molecular InteractionsMolecular Interactions

Prof. Chen Yu ZongProf. Chen Yu Zong

Tel: 6874-6877Tel: 6874-6877Email: Email: yzchen@cz3.nus.edu.sgyzchen@cz3.nus.edu.sg

http://xin.cz3.nus.edu.sghttp://xin.cz3.nus.edu.sgRoom 07-24, level 7, SOC1, NUSRoom 07-24, level 7, SOC1, NUS

22

Biomolecular Interaction: Enzyme + SubstrateBiomolecular Interaction: Enzyme + Substrate

E + S ==> E + P

• This is a generalization of how a biochemist might represent the function of enzymes.

33

Biomolecular Interaction: Enzyme + SubstrateBiomolecular Interaction: Enzyme + Substrate

E + S ==> E + P

kinase-ATP complex + inactive-enzyme ==> Kinase + ADP + active enzyme

K

ATP ADP

P

• Here is an example of the generalization represented by two different ways.

44

Biomolecular Interaction: Enzyme + SubstrateBiomolecular Interaction: Enzyme + Substrate

• This is another representation.

Kinase-ATPcomplex

Activeenzyme

inactiveenzyme

ADP

55

Biomolecular InteractionBiomolecular Interaction

• This is a generalization of the representation.

A B

C D E F…

66

Biomolecular FunctionBiomolecular Function

• A biomolecule’s function can be defined by the things that it interacts with and the new (or altered) molecules that result from that interaction.

A B

C D E F…

77

Biomolecular FunctionBiomolecular Function

• This representation makes it easy to focus on the interaction part.

A B

C D E n…

88

A Simple BIND RecordA Simple BIND Record

• The minimal BIND record has 9 pieces of information.

A B

1. Short label for A 2. Short label for B3. Molecule type for A 4. Molecule type for B5. Database reference for A 6. Database reference for B7. Where A comes from 8. Where B comes from

9. Publication reference

99

An Example BIND RecordAn Example BIND Record

• You can view this record in BIND

A B

1. INAD 2. TRP3. Protein 4. Protein5. GenBank GI 3641615 6. GenBank GI 73018617. GenBank Taxonomy ID 7227 8. GenBank Taxonomy ID 7227

9. PubMed ID 8630257

1010

BIND Stores Molecular Interaction DataBIND Stores Molecular Interaction Data

BIND Interaction Types

Protein - Protein54%

Protein - DNA25%

Other9%

Protein - Not Specified

12%

Protein - RNA1%

Gene - Gene4%

Small Molecule - Gene1%

Protein - Small Molecule

1%

1111

BIND Stores Molecular Interaction DataBIND Stores Molecular Interaction Data

1212

BIND Records are Based on ObservationsBIND Records are Based on Observations

• All BIND records will have a publication reference and most will specifically list a method(s) used to demonstrate the interaction.

A B

1. Short label for A 2. Short label for B3. Molecule type for A 4. Molecule type for B5. Database reference for A 6. Database reference for B7. Where A comes from 8. Where B comes from

9. Publication reference

1313

Methods for Detecting Interactions.Methods for Detecting Interactions.

• A great deal of interaction data in BIND originates from high-throughput experiments designed to detect interactions between proteins.

• The most common methods are:– Two-hybrid assay– Affinity purification

1414

Experimental Evidence of Interaction in BINDExperimental Evidence of Interaction in BINDInteraction Experimental Evidence Captured

Affinity Chromatography

8%

SGA8%

Three Dimensional Structure

20%Cross Linking25%

Two Hybrid Test38%

Other1%

Interaction Experimental Evidence Captured

Other8%

Colocalization1%

Competition Binding

1%

Fluorescence Anisotropy

6%

Not Specified6%

Resonance Energy Transfer

9%

Gel Filtration Chromatography

14%

Gradient Sedimentation

1%

Gel Retardation Assays

1%

Immunostaining 9%

Microarray14%

Light Scattering11%

Electron Microscopy

2%

Elisa6%

Equilibrium Dialysis

16%

Remaining1%

1515

Experimental Method: Two-Hybrid AssayExperimental Method: Two-Hybrid Assay

1.

2. 3.

4.

1616

Experimental Method: Two-Hybrid AssayExperimental Method: Two-Hybrid Assay

1717

Experimental Method: Two-Hybrid AssayExperimental Method: Two-Hybrid Assay

1818

Experimental Method: Experimental Method: Two-Hybrid AssayTwo-Hybrid Assay

1919

Experimental Method: Experimental Method: Two-Hybrid AssayTwo-Hybrid Assay

2020

Two-Hybrid AssayTwo-Hybrid Assay

1.

2. 3.

4.

A

B

Fields S. Song O.Nature. 1989 Jul 20;340(6230):245-6. PMID: 2547163

UASG

GAL4-DBD

SNF1

SNF4

Transcription activation domain

Allows growth on galactoseGAL1

2121

Some Two-Hybrid CaveatsSome Two-Hybrid Caveats

1.

2. 3.

4.

A

Does the DBD-fusion have activity by itself?

2222

Some Two-Hybrid CaveatsSome Two-Hybrid Caveats

1.

2. 3.

4.

A

B

Is the ‘interaction’ mediated by some other protein?

C

2323

Some Two-Hybrid QuestionsSome Two-Hybrid Questions

1.

2. 3.

4.

AB

• Are the proteins expressed?• Are they over-expressed?• Are they in-frame?• Are the interacting domains defined?• Was the observation reproducible?• Was the strength of interaction significant?• Was another method used to back-up the conclusion?• Are the two proteins from the same compartment?

2424

Some Two-Hybrid CaveatsSome Two-Hybrid Caveats

1.

2. 3.

4.

B

A

Is the ‘interaction’ bi-directional?

2525

Experimental Method: Affinity PurificationExperimental Method: Affinity Purification

A

Protein of interestTag modification(e.g. HA/GST/His)

This molecule will bindthe ‘tag’.

2626

Affinity PurificationAffinity Purification

A

The cell

2727

B

Affinity PurificationAffinity Purification

A

The cell

Naturally binding protein

Lots of other untagged proteins

2828

B

Affinity PurificationAffinity Purification

A

Ruptured membranes

Cell extract

2929

B

Affinity PurificationAffinity Purification

A

Untagged proteins go through fastest (flow-through)

3030

B

Affinity PurificationAffinity Purification

A

Tagged complexesare slower and come out later (eluate)

3131

B

Some Questions about Affinity PurificationSome Questions about Affinity Purification

A

• Is the bait protein expressed and in frame?• Is the bait protein observed?• Is the bait protein over-expressed?• Are the interacting domains defined?• Was the observation reproducible?• Was the interactor found in the background?• Was the strength of interaction significant?• Was the interaction saturable?• Was the interactor stoichiometric with the bait protein?• Was another method used to back-up the conclusion?• Was tandem-affinity purification (TAP) used?• Was the interaction shown using an extract or a purified protein?• Is the inverse interaction observable?• Are the two proteins from the same compartment?• Are the two proteins known to be involved in the same process?• Is the interactor likely to be physiologically significant?

3232

B

Some Affinity Purification CaveatsSome Affinity Purification Caveats

A

First and most importantly, this is only a representation of the observation.

You can only tell what proteins are in the eluate; you can’t tell how they are connected to one another.

If there is only one other protein present (B), then its likely that A and B are directly interacting.

But, what if I told you that two other proteins (B and C) were present along with A….

B

A

C

3333

B

Complexes with Unknown Binding TopologyComplexes with Unknown Binding Topology

A

Which of these models is correct?The complex described by this experimental result is said to have an Unknown Topology.

C B

A

C B

A

C

3434

B

Complexes with Unknown StoichiometryComplexes with Unknown Stoichiometry

A

Here’s another possibility?The complex described by this experimental result is also said to have Unknown Stoichiometry.

C

A

3535

High Throughput Data in BINDHigh Throughput Data in BIND

• Affinity purification:Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry (2002). PMID: 11805837

• Two-hybrid:A protein interaction map of Drosophila Melanogaster(2003). PMID: 14605208

• Two-hybrid and Affinity purification:A map of the interactome network of the metazoan C. Elegans (2004). PMID: 14704431

• Data from these examples can be retrieved from BIND using a PMID search.

3636

B

How complex Data are Stored in BINDHow complex Data are Stored in BIND

A ?

?

C?

Three interaction records.

3737

B

How Complex Data are Stored in BINDHow Complex Data are Stored in BIND

A ?

?

C?

A complex record in BINDis simply a collection ofinteraction records.

3838

B

Alternate Representations.Alternate Representations.

A ?

?

C?The matrix model (a clique).

B

A

C

3939

B

Alternate Representations.Alternate Representations.

A ?

?

C?The spoke model.

Which model to use?

B

A

C

4040

Spoke and Matrix ModelsSpoke and Matrix Models Vrp1 (bait), Las17, Rad51, Sla1, Tfp1, Ypt7

SpokeMatrixPossible Actual

Topology

Bader&Hogue Nature Biotech. 2002 Oct 20(10):991-7

Simple model

Intuitive, more accurate, but canmisrepresent

Theoretical max. no. of interactions, but many FPs

4141

A view on real data…matrix modelA view on real data…matrix model

6 redox enzymes

7 redox enzymes

Old yellow enzyme

Function?

Interaction KineticsInteraction Kinetics

E + S ==> E + P

kinase-ATP complex + inactive-enzyme ==> Kinase + ADP + active enzyme

K

ATP ADP

P

4343

Reversibility of Chemical Reactions: Reversibility of Chemical Reactions: EquilibriumEquilibrium

• Chemical reactions are reversible• Under certain conditions (concentration, temperature)

both reactants and products exist together in equilibrium state

H2 2H

4444

Reaction RatesReaction Rates

Net reaction rate = forward rate – reverse rate

• In equilibrium: Net reaction rate = 0• When reactants “just” brought together: Far

from equilibrium, focus only on forward rate• But, same arguments apply to the reverse rate

4545

The Differential Rate LawThe Differential Rate Law

• How does the rate of the reaction depend on concentration? E.g.

3A + 2B C + Drate = k [A]m[B]n

(Specific reaction)

rate constant

Order of reaction

with respect

to A

Order of reaction

with respect

to B

m+n: Overall order of

the reaction

4646

Rate Constants and Reaction OrdersRate Constants and Reaction Orders

• Each reaction is characterized by its own rate constant, depending on the nature of the reactants and the temperature

• In general, the order with respect to each reagent must be found experimentally (not necessarily equal to stoichiometric coefficient)

4747

Elementary Processes and Rate LawsElementary Processes and Rate Laws

• Reaction mechanism: The collection of elementary processes by which an overall reaction occurs

• The order of an elementary process is predictable

Unimolecular A* B K+ [A] First order

Bimolecular A + B C + D K+ [A] [B] Second order

Trimolecular A + B + C D + E K+ [A] [B] [C] Third order

4848

Elementary Processes and Rate LawsElementary Processes and Rate Laws

• Reaction mechanism: The collection of elementary processes by which an overall reaction occurs

• The order of an elementary process is predictable

Unimolecular A* B K+ [A] – K- [B] First order

Bimolecular A + B C + D K+ [A] [B] – K- [C] [D] Second order

TrimolecularA + B + C D + E

K+ [A] [B] [C] – K- [D] [E]

Third order