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).

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2 3

Freeelectron

• (b)Tetrahedralintermediatetransitionstate:enzyme’soxyanionholestabilizessubstrateoxygennegativecharge.

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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.

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Samefigure

• (c)Peptidebondbrokenbyadditionalelectronmovements:

• releaseofoneoftheNH2−P2 reactionproducts

• formationoftheacylenzyme(ESʹcomplex)

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What’snext?

• (d)Solventwateroxygenattackstheacylenzymecarbonylcarbon.

• [IfthepHistoolow:theHis-57sidechainisprotonatedandcannotparticipateincatalysis.]

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‘His’needs‘His’friendtosharetheelectron!But‘O’isabadfriendtoattackthe’C’!

• (e)Formationofasecondtetrahedralintermediate

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Attack

• (f)Additionalelectronmovements:•breaktheSer-195–substratebond(formationoftheEPcomplex)

• (inset)His-57sidechainheldintheproperorientationbyhydrogenbondingtotheAsp-102sidechainfacilitatescatalysisbywithdrawinganddonatingprotonsthroughoutthereaction

• releasetheP1−COOHreactionproduct

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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)

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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.