讲座分析化学发展

—••αββ••¡α¡310¡ββ¡ββ¡SARsPKHIVUnfolding.movPuklin-Faucher.S4.movPuklin-Faucher.S2.mov••••FourierTransformInfraredSpectrumFT-IRααββββ~α~β—C8H10——•••••AbsorbanceAmideI:C=OstretchingAmideII:NHbending•SecondaryDerivativespectrum--C=O---H-O13001400150016001700180019002000AbsorbanceCol21vsCol22Wavenumber(cm-1)160016201640166016801700d2A/dν2AmideIAmideIIWavenumber(cm-1)160016201640166016801700-d2A/dν2β-Turnβ-turnα-helixβ-sheetrandomsidechain•ii+4•α--3.6;310--3;β--2(cm-1)16241.0β16272.0β16332.0β16382.0β16421.0β16482.016562.0α16633.031016671.0β16751.0β16802.0β16852.0β16912.0β16962.0β•Noabsorbanceat2000-1750cm-1•H/Dexchange:AmideI/AmideIIProteinFT-IRSpectrum—TwostandardsEPACH-Dexchange(with300uMcAMP)Wavenumber(cm-1)130014001500160017001800Absorbance0.00.05.10.15.20H2O1'3'5'10'30'60'120'180'D2OFT-IRspectraofPK•SubtractionofH2Owillbringbigerrorinabsorbancespectrum•SmallerrorinsecondderivativespectrumWavenumber(cm-1)1300135014001450150015501600165017001750AbsorbanceWavenumber(cm-1)15801600162016401660168017001720Absorbance/Wavenumber2PKPK-negativeerrorPK-positiveerrorPKspectra1400150016001700180019002000AbsorbanceWavenumber(cm-1)160016201640166016801700d2A/dν2H/DexchangeExchangedTime(min)020406080100120140160180200Ratioofunexchangedproton0.00.05.10.15.20.25.30H/DexchangeWavenumber(cm-1)13001400150016001700180019002000Absorbance1674CRP+0.8MGdnHCl0.8MGdnHClCRP(10x)ABCFT-IRspectra—inthepresenceofdenaturant•Havetogetgoodabsorbancespectrumfirst•Becareful,whensampleinthepresenceofGdnHCl!Questions:•H2O1650cm-1•pathlength•H/Dexchange:AmideI/AmideII•1700-1600cm-1••1700-1600cm-1•absorbanceat2000-1750cm-1?CircularDichroism(CD)•••—•MOS-450•50.5mg/ml1¡¡¡()/()CD••θ·θ·θ·θθθθθ•θ·θ·θ·θθ·θ·θ·θθ·θ·θ·θ••••(k2d,???)•CD…••NearUVCD…CD…CD…•FRET(FluorescenceResonanceEnergyTransfer)FluorescenceResonanceEnergyTransfer(FRET)DonorAcceptorET601⎟⎠⎞⎜⎝⎛=rRkDTτTDTkkE+=-1τRateofenergytransfer(kT)rsteroF..R0isthedistanceatwhichhalfoftheenergyistransferred(20-50Å)66060rRRE+=Efficiencyofenergytransfer(E)τDisthedecaytimeofthedonorintheabsenceofacceptorrsteroF..Theoryr—distancebetweendonorandacceptorExample……..StyrerandHauglandshowedthatFRETcouldbeusedasaspectroscopicrulerFRETisalwaysusedtomeasuredistancesontheorderofR0,Though,itisbettersuitedfordetectingchangesindistance(conformation)ApplicationsGeneticengineeringforprobesandlabelingNaturestructuralbiology,2000(7),9:730-734Strategy•Acceptor•Aonor•Distance—inthepresenceandabsenceofXX1MSEPKAIDPKLSTTDRVVKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHL51MKEGRLEESVALRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMK101LFEVGGSPANTRYLFLGDYVDRGYFSIECVLYLWALKILYPKTLFLLRGN151HECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCVHG201GLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEKTQEHFTH251NTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTGFP301SLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVF351TWSLPFVGEKVTEMLVNVLNICSDDELGSEEDGFDGATAAARKEVIRNKI401RAIGKMARVFSVLREESESVLTLKGLTPTGMLPSGVLSGGKQTLQSATVE451AIEADEAIKGFSPQHKITSFEEAKGLDRINERMPPRRDAMPSDANLNSIN501KALTSETNGTDSNGSNSSNIQCalcineurinACNandCaMConstructs??ThiolModificationFluorescenceSpectraofDonorandacceptorDonorAcceptorThiolModification•50uMproteininBuffer(50mMTris,100mMNaCl,2mMMg2+,20uMTCEP,pH7.5);•NoDTT,littleTCEP;•Oxygenfree(better!);•10-foldexcessofdye;•At4Covernight(!!!)•G-25(twice)toremoveexcessofdye(extensivedialysisagainstbuffer)•Dyecons:Absorbancespectrum;Proteincons:Bradfordassay;•Addbufferintoprotein;•Aliquotat-70C.IFFREThappensbetweenCNandCaM…MeasuringdistancesR6=R06(1-E)/EE—transferredenergy;R—distancebetweendonorandacceptor;τd—lifetimeofthedonorintheabsenceofacceptor;τda—lifetimeofthedonorinthepresenceofacceptor.E=1-τd/τdaR0=9.7x103(Jk2n-4q)1/6(Å)R0—distancebetweendonorandacceptoratwhichthe50%energyistransferred;J—Overlapintegralofabsorbancespectrumandemissionspectrum;n—Assumedtobe1.33;k2—Assumedtobe2/3;q—Assumedtobe1whenthereisnoquenchingbywater.•SEC(Size-ExclusionChromatography)•FullProcessCapability—Fullcapabilityfromsampleinjectionthroughfractioncollection,includingautomatedcolumnwashingandregeneration.•Speed—Chromatographicseparationsinaslittleasonetothreeminutes.•Versatility—Multipleinjectionmodeoptions.•Detection—MonitoringofpH,conductivity,dual-wavelengthUV/Visabsorbance,fluorescence(optional),andsystempressure.•DataAnalysis—Dataanalysisofsingledatafilesorgroupsofdatafiles.cAMPReceptorProtein…Time(Sec)400500600700AbsorbanceH159LD53HHis-CRPL148RK52NWTK52/H159S62FT127LS-CRPConformationalchangeinducedbymutation…Time(Sec)450500550600650AbsorbanceProteinUnfolding…[GdnHCl](M)01234Fraction-.20.0.2.4.6.81.01.2ProteinUnfolding…