Download Macromolecular Interaction

MacromolecularInteraction
AdvancedGenetic
YangLi
Feb.272017
Proteinsformcomplexes/interactwithother
macromolecules
Howcanyouidentifyprotein-protein
interactions?
Howdoyouidentifyprotein-protein
interactions?
Biochemical/biophysicalmethods
• Affinitypurification– massspectrometry
• Co-immunoprecipitation
• Proteinmicroarrays
• Förster resonanceenergytransfer
Genetic/molecularmethods
• Yeast-twohybridsystem
• Split-ubiquitinsystem
• Bacterial-two-hybridsystem
Y2H:usingthesplitGal4-UASsystemtogo
“fishing”forprotein-proteininteractions
ThisishowGal4UASnormallyworks:
UAS=Upstreamactivationsequence
Gal4=atranscriptionfactormadeupofaDNAbindingandanactivation
domain
TheDNAbindingdomainandactivation
domainsofGAL-4canbeseparated
TheDNAbindingdomain(BD)alonecannotactivate
transcription
The“bait”(i.e.yourproteinofinterest)isfusedtothe
BD
TheDNAbindingdomainandactivation
domainsofGAL-4canbeseparated
Theactivationdomain(AD)alonecannotbindtheUAS
The“prey”isfusedtotheAD
Y2H:usingthesplitGal4-UASsystemtogo
“fishing”forprotein-proteininteractions
Ifthe“prey”bindstothe“bait”theADandBDare
broughttogetherandactivatetranscriptionofthe
reporter
andaninteractionisdiscovered.
Y2H:usingthesplitGal4-UASsystemtogo
“fishing”forprotein-proteininteractions
Testmanydifferent“prey”
e.g.cDNAlibrary
CreatingacDNAlibrary
Reporters
• Auxotroph->prototrophselection
• Aminoacidbiosynthesis(His,Leu,Ade,Ura)
• Nucleicacidbiosynthesis
• Colordetectionscreen
• LacZ:blue/whitescreen
• GFP
Yeasttwo-hybridassayofdifferentLexAfusionproteins(baits)withvariousPDZ
domains(preys)
HostOrganism
• Yeast
•
•
•
•
•
Eukaryotic
Hospitableinternalenvironment
Genomesequenced,techniquestomanipulate
Onlyabletodetectinteractionsinthenucleus
Someproteinsaretoxictoyeast
• E.coli
• Highertransformationefficiency
• Donotneedanuclearlocalizationsignal
• Canusestrainswithoutinterferingmethyltransferase activity
• MammalianCells
BenefitsofY2H
• Candetecttransientinteractionsnotfoundinco-IP
• Semi-quantitative(2colorsystem)
• Canbeusedtostudyknowninteractions
• Modifyspecificresidues,observewhetherinteractionismaintained
• Immediateidentificationofthegenethatencodestheproduct
WeaknessesofY2H
• Falsepositives
• Proteinsmaynotbeexpressedtogetherinreality
• Unnaturalconcentrations
• Non-specificinteractions
• Falsenegatives
• Interactionsmustoccurinthenucleus(andproteinsmustbesoluble)tobe
detected
• FusiontoADorBDmayblockproteininteraction
• Yeastmaylackchaperonesforproperfolding
RelatedScreens
• Reverseyeast-twohybrid
• Detectwhenaninteractionisdisrupted
• Yeastthree-hybrid
• DetectProtein-RNAinteractions
• Yeastone-hybrid
• DetectProtein-DNAinteractions
ReverseY2H
Somethingdisruptstheinteraction:
1
Howdoyouknowtheinteractionwas
disrupted?
ReplicaPlating
Howdoyouknowtheinteractionwas
disrupted?
URA3geneand5-fluoro-oroticacid(5-FOA)
Yeastsurviveswhenproteininteractionisdisrupted
Protein-RNAinteractions:
Yeastthree-hybrid
Gal4BD-Protein1-RNA-Protein2-Gal4AD
Protein-DNAinteractions:
Yeast-one-hybrid
Widespread
Macromolecular
Interaction
(Vidaletal.2015)
Experimental
Workflow
LUMIERAssay
MostdiseasemissensemutationsDoNotimpairproteinfoldingorstability
Interactionperturbationprofilesdistinguishdiseasemutationsfromnon-diseasevariants.
Protein-DNAinteractionperturbation