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GBME,SKKUMolecular&CellBiology
H.F.K.
Chapter3-II– ProteinStructureandFunction
Activesiteoftheenzymetrypsin.• Enzymes(proteinsorRNAs)catalyzemakingorbreakingsubstratecovalentbonds.• (a)Trypsin (serineprotease)activesite:
• Substrate-bindingpocket – bindsspecificsubstrate• Catalyticsite– containssidechainsofthecatalytictriadSer-195,Asp-102,andHis-57thatbreakspeptidebonds.
• Insomeenzymes,thecatalyticandsubstrate-bindingsitesoverlap;inothers,thetworegionsarestructurallydistinct.
Schematicmodelofanenzyme’sreactionmechanism.
Free-energyreactionprofilesofuncatalyzedandmultistepenzyme-catalyzedreactions.
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!
Bindingpockets
• Trypsin– bindssubstrate(+)charged arginineandlysinesidechains.• Chymotrypsin– bindslarge,hydrophobicsidechainssuchasphenylalanine.• Elastase– bindssmallsidechainssuchasglycineandalanine.
Howdoestheserineproteasework?
Mechanismofserineprotease–mediatedhydrolysisofpeptidebonds.
• (a)EScomplex:Ser-195hydroxyloxygenattacksthecarbonylcarbonofthesubstrate’stargetedpeptidebond(yellow).
1
2 3
Freeelectron
• (b)Tetrahedralintermediatetransitionstate:enzyme’soxyanionholestabilizessubstrateoxygennegativecharge.
45
4:Nofurtherattack
Oxyanionhole
An oxyanion hole is apocket inthe active site ofan enzyme that stabilizes transition state negative charge ona deprotonated oxygen or alkoxide.
Electronacceptors
• (b)Tetrahedralintermediatetransitionstate:enzyme’soxyanionholestabilizessubstrateoxygennegativecharge.
45
Samefigure
• (c)Peptidebondbrokenbyadditionalelectronmovements:
• releaseofoneoftheNH2−P2 reactionproducts
• formationoftheacylenzyme(ESʹcomplex)
6
What’snext?
• (d)Solventwateroxygenattackstheacylenzymecarbonylcarbon.
• [IfthepHistoolow:theHis-57sidechainisprotonatedandcannotparticipateincatalysis.]
78
‘His’needs‘His’friendtosharetheelectron!But‘O’isabadfriendtoattackthe’C’!
• (e)Formationofasecondtetrahedralintermediate
9
Attack
• (f)Additionalelectronmovements:•breaktheSer-195–substratebond(formationoftheEPcomplex)
• (inset)His-57sidechainheldintheproperorientationbyhydrogenbondingtotheAsp-102sidechainfacilitatescatalysisbywithdrawinganddonatingprotonsthroughoutthereaction
• releasetheP1−COOHreactionproduct
10
Overallreaction
https://www.youtube.com/watch?v=4wZTyVcWfrY
ThepHdependenceofenzymeactivity.
ThinkthefirststepwithHisresidue
pKa =~6.8TrypsinproteaseCytosol:pH~7.2
ProtonatedorNot?Then?
Assemblyofenzymesintoefficientmultienzyme complexes.
Couplingbyascaffoldproteinovercomesslowsubstratediffusioninametabolicpathway
Someenzymesarefusedatthegeneticlevel
ProteinStructureandFunction
3.4RegulatingProteinFunction• Proteinsmayberegulatedatthelevelofproteinsynthesis,proteindegradation,orthroughnoncovalentorcovalentinteractions.
• Proteinsmarkedfordestructionwithapolyubiquitintagbyubiquitinligasesaredegradedinproteasomes.
• Severalallostericmechanismsactasswitches,reversiblyturningproteinactivityonandoff.
• Higher-orderregulationincludestheintracellularcompartmentationofproteins.
Ubiquitin- andproteasome-mediatedproteolysis.
Ubiquitination
• Enzyme+regulatorysubunit
Degradation
Activeenzyme
Hemoglobinbindsoxygencooperatively.
• Tetramerichemoglobin:fouroxygen-bindingsites
• Saturation– allfoursitesloadedwithoxygen.• P50 - pO2 atwhichhalftheoxygen-bindingsitesareoccupied.
• LargechangeinoxygenboundoverasmallrangeofpO2 valuespermitsefficientunloadingofoxygeninperipheraltissuessuchasmuscle.
• Sigmoidalcurve– indicativeofcooperativebinding– bindingofoneoxygenmoleculeallosterically(cooperatively)influencesthebindingofsubsequentoxygens
ConformationalchangesinducedbyCa2+ bindingtocalmodulin.
• Ca2+ bindingchangescalmodulinconformation–hydrophobicsidechainsbecomemoreexposedtosolvent
• Ca2+/calmodulincomplexwrapsaroundconservedsequencesinexposedhelicesofvarioustargetproteins,alteringtheiractivity.
TheGTPase switch.
Regulationofproteinactivitybyphosphorylationanddephosphorylation.
• Kinases:• transferterminalphosphategroupfromATPtospecificS/TorYOHgroups
• activatesomeproteins,inactivateothertypesofprotein
• Mostkinasescanphosphorylatemultipledifferenttargetproteins.
• Phosphatases:• hydrolyzephosphategroupoffprotein–
• inactivatessomeproteins,activatesothertypesofproteins
Practicalproteinworkslearnedbytext
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ProteinStructureandFunction
3.5Purifying,Detecting,andCharacterizingProteins• Proteinscanbeisolated fromothercellcomponentsonthebasisofavarietyofphysicalandchemicalproperties.
• Proteinscanbedetectedandquantifiedbyvariousassaysandspecificantibody recognition.
Weneedcells…
Howtoseparatethespecifictypesofcells?
Thinkthedifferencebetweencells…
Centrifugationtechniquesseparateparticlesthatdifferinmassordensity
Centrifugationtechniquesseparateparticlesthatdifferinmassordensity
Max.80,000rpm!
Boeing747:3,854 rpm
Newtech.tocellseparation…
ContinuousSeparationofMicro-ParticlesbySizeinaCurvedSheathFlow
https://www.youtube.com/watch?v=0VVrt-ge2No
HomogenizationIn cellbiology or molecularbiology research, homogenization isaprocesswherebyabiologicalsampleisbroughttoastatesuchthatallfractionsofthesampleareequalincomposition.
Frenchpress
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.
Thinktheproteinproperties
Weight
Structure
Charge
Whichinformationiseasytouse?
SDS-polyacrylamidegelelectrophoresis(SDS-PAGE)separatesproteinsprimarilyonthebasisoftheirmasses.
sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS)
HowdoesSDSwork?
• sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS)
- -- -
- --
SDS– detergentfordenaturationoftheprotein+DTTorbeta-MeE
SDScoatstheproteinwithauniformnegativecharge.(1.4gSDS/1gprotein)
SDS-PAGE
Thinktheproteinproperties
Weight
Structure
Charge
Isoelectricfocusing
• Isoelectricfocusing (IEF)isanelectrophoretictechniquefortheseparationofproteinsbasedontheir isoelectric point(pI).ThepI isthepHatwhichaproteinhasnonetchargeandthus,doesnotmigratefurtherinanelectricfield.
Two-dimensionalgelelectrophoresisseparatesproteinsonthebasisofchargeandmass.
Twosteps!• Step1:Isoelectricfocusing
• Step2:SDS-PAGEseparatesproteinsingelbasedonmolecularweight.
Two-dimensionalgelelectrophoresisseparatesproteinsonthebasisofchargeandmass.
Findthedifference!
Thenwhatwillyoudo?
Howtodetectthespecificprotein?
Antibody-antigenspecificity
+SDS-PAGE!
Westernblotting
Westernblotting
Toknowthesequence!
Findthedifference!
Getthisprotein
LC-MS?Massspectrometry (MS)isananalyticaltechniquethationizes chemicalspecies andsortsthe ions basedontheir mass-to-chargeratio.
Manhattanproject
Molecularmasscanbedeterminedbymatrix-assistedlaserdesorption/ionizationtime-of-flight(MALDI-TOF)massspectrometry.
• Step1:Pulsesoflaserlightionizeaproteinorpeptidemixturethatisabsorbedonametaltarget(matrix-assisted).
• Step2–3:Anelectricfieldacceleratestheionsinthesampletowardthedetector.
• Thetimeittakesaniontoreachthedetectorisproportionaltothesquarerootofthemass-to-charge(m/z)ratio.
• Results:Smallerionswiththesamechargemovefaster(shortertimetothedetector).
Maldi-TOFvideo!
https://www.youtube.com/watch?v=8R1Oyqx5KfE
Fingerprinting…
• Peptidemassfingerprinting (PMF)(alsoknownas proteinfingerprinting)isananalyticaltechniquefor protein identificationinwhichtheunknownproteinofinterestisfirstcleavedintosmaller peptides,whoseabsolutemassescanbeaccuratelymeasuredwitha massspectrometer suchas MALDI-TOF or ESI-TOF.
Molecularmassofproteinsandpeptidescanbedeterminedbyelectrosprayionizationion-trapmassspectrometry.
• Toppanel:MassspectrumofamixtureofthreemajorandseveralminorpeptidesfromthemouseH-2classIhistocompatibilityantigenQ10αchain.
• TheMS/MSspectrum(product-ionspectrum)providesdetailedstructuralinformation,includingpeptidesequenceinformation.
• peptidesequence(FIIVGYVDDTQFVR)deduction– fromthevaryingsizesoftheproductions,understandingthatpeptidebondsareoftenbrokeninsuchexperiments,knownm/zvaluesforindividualaminoacidfragments,anddatabaseinformation
CombiningwithLC
Threecommonlyusedliquidchromatographic techniquesseparateproteinsonthebasisofmass,charge,oraffinityforaspecificbindingpartner.
InsteadofSDS-PAGE
• Column– porousbeads
• Columns– beadswitheitherapositivecharge(shownhere)oranegativecharge
• Column– beadstowhichaspecificantibodyiscovalentlyattached
LC-MS/MS
LC-MS/MSisusedtoidentifytheproteinsinacomplexbiologicalsample.
• LC-MS/MSidentificationoforganelleproteins:Thinkthestepstoidentifyoneproteinexpressedinaspecificorganelle.
ProteinStructureandFunction
3.6Proteomics• Proteomics– systematicstudyofabundance,modifications,interactions,localization,andfunctionsofallorsubsetsofproteinsinwhole-organism,tissue,cellular,andsubcellularbiologicalsystems.
• Methodsusedforproteomicanalysesinclude2Dgelelectrophoresis,density-gradientcentrifugation,andmassspectrometry.
• Resultsrevealvarioustypesofproteomes.
NextclassCulturingandvisualizingcells
Discussionwithfriends• PleaseexplainthereasonwhylowpHinactivatestrypsinproteasebasedonenzymereaction.
• SeethemostactivepHs oflysosomalhydrolaseandchymotrypsin.HowcanyouexplaintheinvertedU-shapeactivityofeachenzyme.
• Ifoneproteinwasubiquitinated,whatkindofproteinbandchangedoyouseeontheSDS-PAGE?FindonepaperinPubmed site,‘Synapticproteindegradationunderliesdestabilizationofretrievedfearmemory.'
• Pleaseexplainthegraphwithallostericmechanisms
Pubmed sitesearch
참고
pKa ofaminoacids&pI
https://www.youtube.com/watch?v=EH60oHI2wD8
HistidinepKa &pI1
2
3
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.
Disorderedprotein
Intrinsicallydisorderedproteins:mechanismsofbindingtowell-orderedproteinsandidentificationbasedonhydrophobicityandnetcharge.
• Conformationalselection(toppathway):disorderedprotein(PUMA)transientlyadoptsitsboundstatestructure,whichthenbindstothewell-orderedbindingpartner(MLC1).
• Inducedfit(bottompathway):disorderedproteinbeginstobindtothewell-orderedpartnerandthenisinducedtoformtheorderedconformationbycompletingbindinginteractions.
• >30percentofeukaryoticproteinsarepredictedtohaveatleastonedisorderedsegmentof50ormoreconsecutiveresidues.
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