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BIOC23H3 (23)
Lecture

BIOC23: Analysis of protein-protein interactions (word-for-word lec notes)

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Department
Biological Sciences
Course Code
BIOC23H3
Professor
Rongmin Zhao

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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: o AP-MS = affinity purification coupled with mass-spec o Yeast 2-hybrid screening o Protein fragment complementation assay o Peptide array (not just protein-protein interactions; can also be small fragment peptide-protein interactions) • What’s the purpose for determining protein-protein interactions? o 1) 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 o 2) 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 o TAP = tandem affinity purification tag that contains protein A and calmodulin binding peptide = there are 2 affinity purification tags o Ex) 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 o SDS-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? o Can 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) • 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 o When you purify HSP90 by tap-tag, then all those proteins that are bound to HSP90 will be co-purified o At 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 o These 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) o For any gene, ATG promoter; then transcription factor protein with a DBD and AD which will promote RNA polymerase o For 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: o DBD 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 o So 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 o In this case, histidine marker was used; if downstream gene is lacZ; then there’s DNA binding domain attached to B and then another protein fused to the activation domain which is recruited; the AD will trigger the downstream lacZ gene expression and then you can use Blue/White reagent to monitor the interaction o If there’s an interaction, then the activation domain will be recruited to the DNA binding domain which will then trigger the downstream gene expression o If there’s no interaction btn the 2 proteins, there’s no expression of the downstream genes • Principle = simple = just use split transcription factor domains; only when the fused proteins are interacting, the activation domain is recruited to the promoter region and there is downstream expression • Practically speaking there are 2 diff methods: o 1) 2-hybrid screen using an ordered array (one-on-one screen):  Ex) if you want to test if protein A interacts w/ protein B, then can fuse A to DNA binding domain and fuse B to the activation domain; then put into diff mating types and allow them to mate to see if there’s interaction btn the 2 protein types by seeing if the reporter gene is expressed  This is one-on-one b/c you know what the 2 proteins are and you just want to test if they interact  Yeast = 6000 genes; if you want to test if Protein A interacts with all the other proteins or not  put all 6000 genes in the vector and then test them one-by-one  Put onto microplate, each spot represents an individual yeast hybrid clone diploid; then check which proteins can interact with Protein A  Idea is: you want to test if protein A interacts with some protein in
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