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GBME,SKKUMolecular&CellBiology
H.F.K.
Chapter3-II– ProteinStructureandFunction
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Activesiteoftheenzymetrypsin.• Enzymes(proteinsorRNAs)catalyzemakingorbreakingsubstratecovalentbonds.• (a)Trypsin (serineprotease)activesite:
• Substrate-bindingpocket – bindsspecificsubstrate• Catalyticsite– containssidechainsofthecatalytictriadSer-195,Asp-102,andHis-57thatbreakspeptidebonds.
• Insomeenzymes,thecatalyticandsubstrate-bindingsitesoverlap;inothers,thetworegionsarestructurallydistinct.
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Schematicmodelofanenzyme’sreactionmechanism.
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Free-energyreactionprofilesofuncatalyzedandmultistepenzyme-catalyzedreactions.
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Substratebindingintheactivesiteoftrypsin-likeserineproteases.• (a)Trypsinactivesite:
• Substrateformsatwo-strandedβsheetwithtrypsin’ssubstrate-bindingsite.
• Trypsinbindingsitepocketbindssubstratearginine(R3)sidechain–
• EnzymeAsp-189negativechargestabilizessubstrateArg positivelychargedguanidinium group.
• Bindingalignsthesubstrateargininepeptidebondforhydrolysiscatalyzedbytheenzyme’sactive-sitecatalytictriad(sidechainsofSer-195,His-57,andAsp-102).
• (b)Bindingpocket substratespecificity:
Therealphysicalbindingsiteandpocket!
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Bindingpockets
• Trypsin– bindssubstrate(+)charged arginineandlysinesidechains.• Chymotrypsin– bindslarge,hydrophobicsidechainssuchasphenylalanine.• Elastase– bindssmallsidechainssuchasglycineandalanine.
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Howdoestheserineproteasework?
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Mechanismofserineprotease–mediatedhydrolysisofpeptidebonds.
• (a)EScomplex:Ser-195hydroxyloxygenattacksthecarbonylcarbonofthesubstrate’stargetedpeptidebond(yellow).
1
2 3
Freeelectron
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• (b)Tetrahedralintermediatetransitionstate:enzyme’soxyanionholestabilizessubstrateoxygennegativecharge.
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4:Nofurtherattack
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Oxyanionhole
An oxyanion hole is apocket inthe active site ofan enzyme that stabilizes transition state negative charge ona deprotonated oxygen or alkoxide.
Electronacceptors
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• (b)Tetrahedralintermediatetransitionstate:enzyme’soxyanionholestabilizessubstrateoxygennegativecharge.
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Samefigure
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• (c)Peptidebondbrokenbyadditionalelectronmovements:
• releaseofoneoftheNH2−P2 reactionproducts
• formationoftheacylenzyme(ESʹcomplex)
6
What’snext?
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• (d)Solventwateroxygenattackstheacylenzymecarbonylcarbon.
• [IfthepHistoolow:theHis-57sidechainisprotonatedandcannotparticipateincatalysis.]
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‘His’needs‘His’friendtosharetheelectron!But‘O’isabadfriendtoattackthe’C’!
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• (e)Formationofasecondtetrahedralintermediate
9
Attack
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• (f)Additionalelectronmovements:•breaktheSer-195–substratebond(formationoftheEPcomplex)
• (inset)His-57sidechainheldintheproperorientationbyhydrogenbondingtotheAsp-102sidechainfacilitatescatalysisbywithdrawinganddonatingprotonsthroughoutthereaction
• releasetheP1−COOHreactionproduct
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Overallreaction
https://www.youtube.com/watch?v=4wZTyVcWfrY
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ThepHdependenceofenzymeactivity.
ThinkthefirststepwithHisresidue
pKa =~6.8TrypsinproteaseCytosol:pH~7.2
ProtonatedorNot?Then?
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Assemblyofenzymesintoefficientmultienzyme complexes.
Couplingbyascaffoldproteinovercomesslowsubstratediffusioninametabolicpathway
Someenzymesarefusedatthegeneticlevel
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ProteinStructureandFunction
3.4RegulatingProteinFunction• Proteinsmayberegulatedatthelevelofproteinsynthesis,proteindegradation,orthroughnoncovalentorcovalentinteractions.
• Proteinsmarkedfordestructionwithapolyubiquitintagbyubiquitinligasesaredegradedinproteasomes.
• Severalallostericmechanismsactasswitches,reversiblyturningproteinactivityonandoff.
• Higher-orderregulationincludestheintracellularcompartmentationofproteins.
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Ubiquitin- andproteasome-mediatedproteolysis.
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Ubiquitination
• Enzyme+regulatorysubunit
Degradation
Activeenzyme
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Hemoglobinbindsoxygencooperatively.
• Tetramerichemoglobin:fouroxygen-bindingsites
• Saturation– allfoursitesloadedwithoxygen.• P50 - pO2 atwhichhalftheoxygen-bindingsitesareoccupied.
• LargechangeinoxygenboundoverasmallrangeofpO2 valuespermitsefficientunloadingofoxygeninperipheraltissuessuchasmuscle.
• Sigmoidalcurve– indicativeofcooperativebinding– bindingofoneoxygenmoleculeallosterically(cooperatively)influencesthebindingofsubsequentoxygens
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ConformationalchangesinducedbyCa2+ bindingtocalmodulin.
• Ca2+ bindingchangescalmodulinconformation–hydrophobicsidechainsbecomemoreexposedtosolvent
• Ca2+/calmodulincomplexwrapsaroundconservedsequencesinexposedhelicesofvarioustargetproteins,alteringtheiractivity.
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TheGTPase switch.
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Regulationofproteinactivitybyphosphorylationanddephosphorylation.
• Kinases:• transferterminalphosphategroupfromATPtospecificS/TorYOHgroups
• activatesomeproteins,inactivateothertypesofprotein
• Mostkinasescanphosphorylatemultipledifferenttargetproteins.
• Phosphatases:• hydrolyzephosphategroupoffprotein–
• inactivatessomeproteins,activatesothertypesofproteins
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Practicalproteinworkslearnedbytext
��� �������나중에실험도해보시는것이…
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ProteinStructureandFunction
3.5Purifying,Detecting,andCharacterizingProteins• Proteinscanbeisolated fromothercellcomponentsonthebasisofavarietyofphysicalandchemicalproperties.
• Proteinscanbedetectedandquantifiedbyvariousassaysandspecificantibody recognition.
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Weneedcells…
Howtoseparatethespecifictypesofcells?
Thinkthedifferencebetweencells…
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Centrifugationtechniquesseparateparticlesthatdifferinmassordensity
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Centrifugationtechniquesseparateparticlesthatdifferinmassordensity
Max.80,000rpm!
Boeing747:3,854 rpm
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Newtech.tocellseparation…
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ContinuousSeparationofMicro-ParticlesbySizeinaCurvedSheathFlow
https://www.youtube.com/watch?v=0VVrt-ge2No
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HomogenizationIn cellbiology or molecularbiology research, homogenization isaprocesswherebyabiologicalsampleisbroughttoastatesuchthatallfractionsofthesampleareequalincomposition.
Frenchpress
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Sonication!
Cells
Sonication is the act of applying sound energy to agitate particles in a sample, for various purposes. Ultrasonic frequencies (>20 kHz) are usually used, leading to the process also being known as ultrasonication or ultra-sonication.
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Thinktheproteinproperties
Weight
Structure
Charge
Whichinformationiseasytouse?
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SDS-polyacrylamidegelelectrophoresis(SDS-PAGE)separatesproteinsprimarilyonthebasisoftheirmasses.
sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS)
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HowdoesSDSwork?
• sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS)
- -- -
- --
SDS– detergentfordenaturationoftheprotein+DTTorbeta-MeE
SDScoatstheproteinwithauniformnegativecharge.(1.4gSDS/1gprotein)
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SDS-PAGE
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Thinktheproteinproperties
Weight
Structure
Charge
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Isoelectricfocusing
• Isoelectricfocusing (IEF)isanelectrophoretictechniquefortheseparationofproteinsbasedontheir isoelectric point(pI).ThepI isthepHatwhichaproteinhasnonetchargeandthus,doesnotmigratefurtherinanelectricfield.
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Two-dimensionalgelelectrophoresisseparatesproteinsonthebasisofchargeandmass.
Twosteps!• Step1:Isoelectricfocusing
• Step2:SDS-PAGEseparatesproteinsingelbasedonmolecularweight.
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Two-dimensionalgelelectrophoresisseparatesproteinsonthebasisofchargeandmass.
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Findthedifference!
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Thenwhatwillyoudo?
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Howtodetectthespecificprotein?
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Antibody-antigenspecificity
+SDS-PAGE!
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Westernblotting
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Westernblotting
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Toknowthesequence!
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Findthedifference!
Getthisprotein
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LC-MS?Massspectrometry (MS)isananalyticaltechniquethationizes chemicalspecies andsortsthe ions basedontheir mass-to-chargeratio.
Manhattanproject
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Molecularmasscanbedeterminedbymatrix-assistedlaserdesorption/ionizationtime-of-flight(MALDI-TOF)massspectrometry.
• Step1:Pulsesoflaserlightionizeaproteinorpeptidemixturethatisabsorbedonametaltarget(matrix-assisted).
• Step2–3:Anelectricfieldacceleratestheionsinthesampletowardthedetector.
• Thetimeittakesaniontoreachthedetectorisproportionaltothesquarerootofthemass-to-charge(m/z)ratio.
• Results:Smallerionswiththesamechargemovefaster(shortertimetothedetector).
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Maldi-TOFvideo!
https://www.youtube.com/watch?v=8R1Oyqx5KfE
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Fingerprinting…
• Peptidemassfingerprinting (PMF)(alsoknownas proteinfingerprinting)isananalyticaltechniquefor protein identificationinwhichtheunknownproteinofinterestisfirstcleavedintosmaller peptides,whoseabsolutemassescanbeaccuratelymeasuredwitha massspectrometer suchas MALDI-TOF or ESI-TOF.
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Molecularmassofproteinsandpeptidescanbedeterminedbyelectrosprayionizationion-trapmassspectrometry.
• Toppanel:MassspectrumofamixtureofthreemajorandseveralminorpeptidesfromthemouseH-2classIhistocompatibilityantigenQ10αchain.
• TheMS/MSspectrum(product-ionspectrum)providesdetailedstructuralinformation,includingpeptidesequenceinformation.
• peptidesequence(FIIVGYVDDTQFVR)deduction– fromthevaryingsizesoftheproductions,understandingthatpeptidebondsareoftenbrokeninsuchexperiments,knownm/zvaluesforindividualaminoacidfragments,anddatabaseinformation
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CombiningwithLC
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Threecommonlyusedliquidchromatographic techniquesseparateproteinsonthebasisofmass,charge,oraffinityforaspecificbindingpartner.
InsteadofSDS-PAGE
• Column– porousbeads
• Columns– beadswitheitherapositivecharge(shownhere)oranegativecharge
• Column– beadstowhichaspecificantibodyiscovalentlyattached
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LC-MS/MS
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LC-MS/MSisusedtoidentifytheproteinsinacomplexbiologicalsample.
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• LC-MS/MSidentificationoforganelleproteins:Thinkthestepstoidentifyoneproteinexpressedinaspecificorganelle.
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ProteinStructureandFunction
3.6Proteomics• Proteomics– systematicstudyofabundance,modifications,interactions,localization,andfunctionsofallorsubsetsofproteinsinwhole-organism,tissue,cellular,andsubcellularbiologicalsystems.
• Methodsusedforproteomicanalysesinclude2Dgelelectrophoresis,density-gradientcentrifugation,andmassspectrometry.
• Resultsrevealvarioustypesofproteomes.
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NextclassCulturingandvisualizingcells
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Discussionwithfriends• PleaseexplainthereasonwhylowpHinactivatestrypsinproteasebasedonenzymereaction.
• SeethemostactivepHs oflysosomalhydrolaseandchymotrypsin.HowcanyouexplaintheinvertedU-shapeactivityofeachenzyme.
• Ifoneproteinwasubiquitinated,whatkindofproteinbandchangedoyouseeontheSDS-PAGE?FindonepaperinPubmed site,‘Synapticproteindegradationunderliesdestabilizationofretrievedfearmemory.'
• Pleaseexplainthegraphwithallostericmechanisms
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Pubmed sitesearch
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참고
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pKa ofaminoacids&pI
https://www.youtube.com/watch?v=EH60oHI2wD8
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HistidinepKa &pI1
2
3
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A.AspKa valuesAminoacid pKa1 pKa2 pKa3 pI
Glycine 2.34 9.60 --- 5.97
Alanine 2.34 9.69 --- 6.00
Valine 2.32 9.62 --- 5.96
Leucine 2.36 9.60 --- 5.98
Isoleucine 2.36 9.60 --- 6.02
Methionine 2.28 9.21 --- 5.74
Proline 1.99 10.60 --- 6.30
Phenylalanine 1.83 9.13 --- 5.48
Tryptophan 2.83 9.39 --- 5.89
Asparagine 2.02 8.80 --- 5.41
Glutamine 2.17 9.13 --- 5.65
Serine 2.21 9.15 --- 5.68
Threonine 2.09 9.10 --- 5.60
Tyrosine 2.20 9.11 --- 5.66
Cysteine 1.96 8.18 --- 5.07
Asparticacid 1.88 9.60 3.65 2.77
Glutamicacid 2.19 9.67 4.25 3.22
Lysine 2.18 8.95 10.53 9.74
Arginine 2.17 9.04 12.48 10.76
Histidine 1.82 9.17 6.00 7.59
•The pKa values and the isoelectronic point, pI, are given below for the 20 a-amino acids.•pKa1= a-carboxyl group, pKa2 = a-ammonium ion, and pKa3 = side chain group.
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Disorderedprotein
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Intrinsicallydisorderedproteins:mechanismsofbindingtowell-orderedproteinsandidentificationbasedonhydrophobicityandnetcharge.
• Conformationalselection(toppathway):disorderedprotein(PUMA)transientlyadoptsitsboundstatestructure,whichthenbindstothewell-orderedbindingpartner(MLC1).
• Inducedfit(bottompathway):disorderedproteinbeginstobindtothewell-orderedpartnerandthenisinducedtoformtheorderedconformationbycompletingbindinginteractions.
• >30percentofeukaryoticproteinsarepredictedtohaveatleastonedisorderedsegmentof50ormoreconsecutiveresidues.
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