哈佛分子生物学-蛋白质和DNA的关系课件

Protein-DNAInteractionsEquilibriaandfreeenergiesEstimatebindingsiteoccupancyForcesdrivingprotein-DNAinteractionsLambdarepressorMethodstostudyprotein-DNAinteractionsProtein-DNAInteractionsEquili1AAAADDAAAADMH1AAAADDAAAADMH12AADMMajorA-TAADMT-AMinorA-TT-AAAAAAADHG-CC-GG-CC-GMajorHDAAAADAADMinorThe4MajorGroovePatternsAreDistinct2AADMMajorA-TAADMT-AMinorA-TT-A3AAAADDAAAADMH1HyrdophobicGroupsonBasesAAAADDAAAADMH1HyrdophobicGrou43Helix-Turn-HelixMotif34Aº3Helix-Turn-HelixMotif34Aº534125N-term3’434125N-term3’46SpecificityDeterminants?GenericInteractions?5SpecificityDeterminants?Gener76>104>1036>104>1038Gln33NetworkswithGln44Ser∆∆G=4.5kcalLeu∆∆G=>6.2kcal7Gln33NetworkswithGln44Ser∆9ConclusionDNAbindingspecificityfrommajorgrooveinteractionsMutationinoneAAaffectsneighbors-->Protein-DNAinterfacescontainanintricateNetworkofhydrogenbondsConclusionDNAbindingspecific10BDNA434Complex94basesdon’tcontactproteinButveryImportant!BDNA434Complex94basesdon’t11FilterBindingvacuum%BoundProtein[nM]02040608010010050Kd=20nM10Filtervacuum%BoundProtein[12Immunoprecipitaiton%BoundProtein[nM]02040608010010050Kd=20nM10ASepharoseImmunoprecipitaiton%BoundProt13Gel-Shift%ShiftedProtein[nM]02040608010010050Kd=20nM[Protein]11Gel-Shift%ShiftedProtein[nM14DNAseIFootprinting%ProtectedProtein[nM]02040608010010050Kd=20nM[Protein]+DNAseI12DNAseI%ProtectedProtein[nM15CooperativeDNAbindingBytherepressor13CooperativeDNAbinding1316InterferenceFootprintingDNA+MMS+Saturating[Protein]GelShift14InterferenceFootprintingDNA+17ElectronMicroscopy15ElectronMicroscopy1518YFPTilt+RollTilt-Roll19TiltRollYFPTilt+RollTilt-Roll19TiltRol19InteractionofTBP(TATABindingProtein)WithDNAMajorGrooveMinorGroove17InteractionofTBP(TATABindi20Protein-InducedDNABendingFromPositiveRoll185’3’Protein-InducedDNABendingFr21Pyrimidine-PurineStepsHaveLittleBaseStacking5’3’3’5’CGAT19A(Lecture1)Pyrimidine-PurineStepsHaveL22Foradvancedreading:

NucleicAcids:

Structures,Properties,andFunctions

V.A.Bloomfield

D.M.Crothers

I.TinocoForadvancedreading:

Nucleic238Gln∆∆G=5.2kcalLys∆∆G=4.1kcal8Gln∆∆G=Lys∆∆G=24Protein-DNAInteractionsEquilibriaandfreeenergiesEstimatebindingsiteoccupancyForcesdrivingprotein-DNAinteractionsLambdarepressorMethodstostudyprotein-DNAinteractionsProtein-DNAInteractionsEquili25AAAADDAAAADMH1AAAADDAAAADMH126AADMMajorA-TAADMT-AMinorA-TT-AAAAAAADHG-CC-GG-CC-GMajorHDAAAADAADMinorThe4MajorGroovePatternsAreDistinct2AADMMajorA-TAADMT-AMinorA-TT-A27AAAADDAAAADMH1HyrdophobicGroupsonBasesAAAADDAAAADMH1HyrdophobicGrou283Helix-Turn-HelixMotif34Aº3Helix-Turn-HelixMotif34Aº2934125N-term3’434125N-term3’430SpecificityDeterminants?GenericInteractions?5SpecificityDeterminants?Gener316>104>1036>104>10332Gln33NetworkswithGln44Ser∆∆G=4.5kcalLeu∆∆G=>6.2kcal7Gln33NetworkswithGln44Ser∆33ConclusionDNAbindingspecificityfrommajorgrooveinteractionsMutationinoneAAaffectsneighbors-->Protein-DNAinterfacescontainanintricateNetworkofhydrogenbondsConclusionDNAbindingspecific34BDNA434Complex94basesdon’tcontactproteinButveryImportant!BDNA434Complex94basesdon’t35FilterBindingvacuum%BoundProtein[nM]02040608010010050Kd=20nM10Filtervacuum%BoundProtein[36Immunoprecipitaiton%BoundProtein[nM]02040608010010050Kd=20nM10ASepharoseImmunoprecipitaiton%BoundProt37Gel-Shift%ShiftedProtein[nM]02040608010010050Kd=20nM[Protein]11Gel-Shift%ShiftedProtein[nM38DNAseIFootprinting%ProtectedProtein[nM]02040608010010050Kd=20nM[Protein]+DNAseI12DNAseI%ProtectedProtein[nM39CooperativeDNAbindingBytherepressor13CooperativeDNAbinding1340InterferenceFootprintingDNA+MMS+Saturating[Protein]GelShift14InterferenceFootprintingDNA+41ElectronMicroscopy15ElectronMicroscopy1542YFPTilt+RollTilt-Roll19TiltRollYFPTilt+RollTilt-Roll19TiltRol43InteractionofTBP(TATABindingProtein)WithDNAMajorGrooveMinorGroove17InteractionofTBP(TATABindi44Protein-InducedDNABendingFromPositiveRoll185’3’Protein-InducedDNABendingFr45Pyrimidine-PurineStepsHaveLittleBaseStacking5’3’3’5’CGAT19A(Lecture