BIOC23H3 Lecture Notes - Beta Barrel, Methionine, Ubiquitin

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5 Apr 2012
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Lec 12: Analysis of protein-protein interactions methods to determine protein-
protein interactions =
AP-MS
Yeast two-hybrid screening
Protein fragment complementation
Peptide array
Slide 5:
Mentioned b4 that size exclusion chromatography can be used to determine protein-
protein interactions
Other methods for the analysis of protein-protein interactions:
oAP-MS = affinity purification coupled with mass-spec
oYeast 2-hybrid screening
oProtein fragment complementation assay
oPeptide array (not just protein-protein interactions; can also be small
fragment peptide-protein interactions)
What’s the purpose for determining protein-protein interactions?
o1) investigate the interaction btn 2 intrinsic proteins; for ex, if you know
that there are 2 proteins of interest, A & B, and you want to test if these 2
proteins can interact with each other in cell 1:1
o2) screen potential interactors for an interest protein; for ex, if for one
protein, want to find out what are the proteins inside the cell that can
potentially interact with this protein screening method 1:all
Slide 6/7: AP-MS = affinity purification couples with mass spec
Affinity purification where tap-tag used
oTAP = tandem affinity purification tag that contains protein A and
calmodulin binding peptide = there are 2 affinity purification tags
oEx) if HSP90 is bound by calmodulin binding peptide and then protein A;
when you mix the cell lysate and put it thru IgG resin; protein A will bind
the IgG; after cleavage is applied to the calmodulin, will get the protein of
interest that’s bound the calmodulin binding site run this protein on
regular SDS-PAGE
oSDS-PAGE: see molecular marker; will get nothing if HSP90 is not tap-
tagged (lane 2); if HSP90 is tap-tagged, then will get HSP90 band (approx
90 kDa) and will also get many other bands of diff sizes How to
determine what these proteins are?
oCan analyze this sample using tandem mass spec
Have to digest with trypsin and then apply to mass spec
Find out the sequence in mixture and then search database; at the
end, will identify several proteins in the sample
These proteins = likely HSP90 interactors (at least candidates)
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Idea is that you can always use affinity purification to purify HSP90; however,
when you purify the proteins, its in the physiological condition (usu pH/salt); this
condition will preserve protein-protein interaction
oWhen you purify HSP90 by tap-tag, then all those proteins that are bound to
HSP90 will be co-purified
oAt the end, can use mass spec to identify all the peptides that are co-purified
with HSP90; potential interactors with HSP90
Key idea = co-purification to preserve protein-protein interaction during the affinity
purification = use physiological conditions; don’t use disruptive reagents (SDS/salt)
Slide 8: Yeast-2 hybrid Properties of yeast cell and genetics
Need special type of yeast = baking yeast = budding duplication
First euk organism who was sequenced = 16 pairs of chromosomes = diploid;
haploid = 16 individual chromosomes
Contains 6000 open reading frames (aka: 6000 protein coding genes); most yeast
genes (open reading frames) don’t contain introns (unlike mammalian/plant cells);
therefore very easy, whenever cloning genetic sequence
Advantage of this yeast is that there are 2 mating types: MATα and MATa = kind
of like male/female gametes
oThese 2 mating types grow equally well as diploid; haploid grows equally
well as diploid although haploid may be a bit smaller
For yeast-2 hybrid, use the property that haploid and diploid grow very well; then
can introduce the plasmid in the haploid, in 2 diff mating types; then put the 2
mating types, under certain conditions the 2 mating types will mate = fuse to
make diploid; after fusion = get 16 pairs of chromosomes; the 2 nuclei fuse and the
cytoplasm also mix; can bring 2 genes into the same cell why it’s called yeast-2
hybrid
Slide 9: Yeast 2-hybrid Screen Principle
Yeast 2-hybrid uses the property of split transcription factor; transcription factor
usu contains 2 domains = one is DNA binding domain (DBD) and the other is
transactivation domain (AD)
oFor any gene, ATG promoter; then transcription factor protein with a DBD
and AD which will promote RNA polymerase
oFor most transcription factors, can separate the DBD and AD into 2 parts
this is 2 parts of the original transcription factor; put these 2 parts on diff
proteins of interest; put the diff domains into diff mating types of the cell
Diagram:
oDBD bound to B and AD bound to ORF which are found in 2 diff cells;
after mating these 2 diff cells together = fuse to form diploid and the 2
plasmids are now part of the same cell
oSo if the protein binds to the B, b/c activation domain will be recruited to
the place of the DNA binding domain; then AD will trigger the downstream
gene transcription/expression this is the idea of yeast 2- hybrid
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