biomolecular interaction: enzyme + substrate
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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
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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.
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Biomolecular Interaction: Enzyme + SubstrateBiomolecular Interaction: Enzyme + Substrate
• This is another representation.
Kinase-ATPcomplex
Activeenzyme
inactiveenzyme
ADP
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Biomolecular InteractionBiomolecular Interaction
• This is a generalization of the representation.
A B
C D E F…
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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…
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Biomolecular FunctionBiomolecular Function
• This representation makes it easy to focus on the interaction part.
A B
C D E n…
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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
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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
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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
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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
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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
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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%
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Experimental Method: Two-Hybrid AssayExperimental Method: Two-Hybrid Assay
1.
2. 3.
4.
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Experimental Method: Two-Hybrid AssayExperimental Method: Two-Hybrid Assay
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Experimental Method: Two-Hybrid AssayExperimental Method: Two-Hybrid Assay
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Experimental Method: Experimental Method: Two-Hybrid AssayTwo-Hybrid Assay
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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
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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
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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?
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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’.
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Affinity PurificationAffinity Purification
A
The cell
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B
Affinity PurificationAffinity Purification
A
The cell
Naturally binding protein
Lots of other untagged proteins
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B
Affinity PurificationAffinity Purification
A
Ruptured membranes
Cell extract
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B
Affinity PurificationAffinity Purification
A
Untagged proteins go through fastest (flow-through)
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B
Affinity PurificationAffinity Purification
A
Tagged complexesare slower and come out later (eluate)
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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?
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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
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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
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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
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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.
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B
How complex Data are Stored in BINDHow complex Data are Stored in BIND
A ?
?
C?
Three interaction records.
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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
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B
Alternate Representations.Alternate Representations.
A ?
?
C?The spoke model.
Which model to use?
B
A
C
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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
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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)
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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
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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
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