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CZ5211 Topics in Computational BiologyCZ5211 Topics in Computational Biology
• 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%
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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
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?
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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?
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.