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uploaded by user Yuyumon Class: Lecture/Exam: School: Semester: Professor: BIO 361 Exam 1, Lectures 1-13 SBU Fall 2013 Steven Glynn

BIO361EXAM1Lectures113

1stLectureMondayAug26th,2013Thermodynamics Thermodynamics Thiscoursewilldiscuss:Howsimplephysicallawscanbeappliedtobiologicalsystems Question:Whatisthermodynamics? Answer:Dealswithhowenergy,workandheatrelatetoeachother.Definesitsrelationship Lifeobeyslawsofthermodynamics.Thisisthestudyofbiochem. Terms Energy Work

Isthecapacityofasystemtodowork.Transferheat. ORDERED:transferofenergyfromsystemtosurroundingstodosomething useful Heat UNORDERED:transferofenergyduetotemperaturedifference.Unordered becauseitisrandommolecularmotionfromsystemtosurrounding. System Inbiochem:Yourmolecule,proteinetc. Surrounding Everythingelse

Firstlawofthermodynamicsconservationofenergy Definition:Energyofisolatedsystemmustremainconstant.Internalenergyisthetotalenergy containedinsystem.Internalenergycanchangeifeitherheatorworkistransferredwith surrounding.Changeininternalenergy=transferofenergyasheatorwork.Anytransferhasto beheatorworktransferredinorout

U(Energy)=UfinalUinitial=q(heat)w(work)

Equationabove:Explainswhyperpetualmotionsystemdoesntwork.Needtocreateenergyin systemtocompensateforenergytransferredthroughheatorwork. Biologicalsystems:Constantpressure.Alwayssameasoutsideenvironment Enthalpy Definition:Internalenergyofsystem+theworkrequiredtomakeroomforitbydisplacing environment. Preferredexpressionofsystemchangesinchemistryandbiology. Atconstantpressure(biologicalsystems)energyistransferredtosurroundingthrough expandingorheating.Scienceneedednewthermodynamicquantitytotakethatintoaccount Enthalpy. H(enthalpy)=U+P(pressure)V(volume) measuredinjoules(J)

since:H(enthalpy)=q(heat)w(work)+PVandPV=w =>H=qp(heatatconstantpressure) H=10kJ=releaseof10kJofheat Atconstantpressure,enthalpyisequivalenttoheat.Thisisthecaseinmostbiochemical systems.Changeofenthalpyisequivalenttoheat. Sincevolumechangesinbiologicalsystemsaresmall,wecanneglectPVandtherefore H=Uandtheenergychangeofasystemisequivalenttoitsenthalpychange,orheatlossor gain. Spontaneousprocesses Processthatoccurwithoutinputofenergy.Releasesenergytobringsystemintoalower,more thermodynamicallystableenergystate.Thismeansthesystemgoesfromordertomore disorder. ex.Diffusion Nonspontaneousprocess Donthappenbythemselves.Needinputofenergy. Processesthathappenbythemselvesincreasedisorder Processesthatdonthappenbythemselvesdecreaseorder Secondlawofthermodynamics Theentropyofanisolatedsystemneverdecreases,butincreases.Systemsevolvetoastateof thermodynamicequilibriumstateofmaximumentropy. Entropy(S) Numberofdifferentwaysonecanarrangeasystemtostillbethermodynamicallyequivalentto eachother.Oritcanbeseenasthesystemsdisorder.Degreeofrandomness.Entropyofa systemdependsontherandomness,ordisorder,orinotherwordsconcentration.
Ssystem+Ssurrounding=Suniverse>0

Equationstates:Ifaspontaneousprocesshasnooverallchangeinenergyorenthalpy,the changeinentropymustbegreaterthanzeroEntropyoftheUniverseincreases GibbsFreeEnergy Determinedbythesystemsentropyandenthalpychange.

S>H/T

Relationofentropytoenthalpy Criteriaforaspontaneousprocess G=HTS EquationGibbsFreeEnergy IfG<0theprocessisspontaneously(exergonic)

HTS<0

IfG>0theprocessisnonspontaneous(endergonic) IfG=0theprocessisinequilibrium Willthereactionbespontaneousornot?

H S G=HTS

Enthalpicallyfavored(exothermic)andentropicallyfavored. >Spontaneous(exergonic)atalltemperatures. Enthalpically(exothermic)favored(exothermic)butentropically not. >Spontaneous(exergonic)(exergonic)attemperaturesbelow T=H/S Enthalpicallynotfavored(endothermic)butentropicallyfavored. Spontaneous(exergonic)attemperaturesabove T=H/SEnthalpicallynotfavored(endothermic,entropically favored. >Spontaneous(exergoniconlyattemperaturesoverT=H/S Enthalpically(endothermic)andentropicallynotfavored. >Nonspontaneous(endergonic)atalltemperatures Enthalpically(endothermic)andentropicallynotfavored. >Nonspontaneous(endergonic)atalltemperatures.

Anonspontaneouscanhappen,butithastobedrivenbyspontaneousreaction. Entropychangeofreactionwilldependonconcentrationofreactants. Concentrationincrease=Entropydecrease Concentrationdecrease=Entropyincrease Ceffectsreactionentropy. Concentrationincrease=Entropydecrease Concentrationdecrease=Entropyincrease GibbsFreeEnergy TheGibbsfreeenergychangeofareactiondependsontheconcentrationofthe reactants/products.
G=standardfreeenergychange(staysstaysconstant).Alsoreferencestate G=G+RTln([C]^c[D]^d/[A]^a[B]^b)

Equationstates:Thefreeenergychangeofareactiondependsontheconcentrationofthe reactants/products Equilibrium Reactionisatastateinwhichtheforwardreactionandthereversereactionhappenatthesame

rate.AtaconsequencethereactionisbalancedandtheGibbsfreeenergychange=0. Gdegree=RTlnKeq(equilibriumconstant) Keq=e^(Gdegree/RT) ln*Keq=(Hdegree/R)*(1/T)+(Sdegree/R) GasconstantR=8.3145JK^1*mol^1

GGivenstandardfreeenergydatawecandeterminewherethereactionwillbeatequilibrium

StandardState Referencestate(Ex:Sealevel=0) Forreactions:StandardTempequals25celsius,pressure1atm,solutehasactivity=1. Statefunctions Gibbsfreeenergy,energyU,enthalpyH,andentropyarestatefunctions. Statefunctionisthingswhichvaluesonlydependontheircurrentstate,nothowtheygotthere. Thereforeonlychangesinthesestatesareimportantforthermodynamicsandonlythesecanbe measured. Biochemistrystateconventionsstateconventions A)WateractivityisWateractivity1,sowecanbeignoredinreactions. B)HydrogenionactivityisHydrogenionactivity1atpH7 C)Inacidbasereactions:Standardstateofasubstancereactingisdefinedastheconcentration ofthereactionmixtureatpH7. Life Lifeinastateoflowentropy,highlystructured.Accordingtothetriestostayinalowentropy level.Itdecreasesitsownentropylevelbyincreasingtheentropyofitssurroundings,or disorderingit,thereforestillincreasingentropyoverall.Thereforelifestillobeyssecondlawof thermodynamics. Alloflifesreactionsmusthaveaentropyraisingreactionlinkedtoanentropyloweringreaction. Thismeansthatforeveryreactiontocreatecomplexmoleculesorgivemoleculesorder,there mustbeareactioncreatinghighertotallevelsofentropy. Food=highenthalpy,lowentropy Waste=lowenthalpy,highentropy

Lifetriestolowerentropyandthereforepreventtriestopreventbeingatotalequilibrium. Weneedareactionthatcreatesanincreaseofentropytolowerourownentropy.

Enzymes

Enzymesarecatalystsforbiochemicalreactions,byprovidinganenergeticallymorefavorable pathwayforthereactiontoproceed. Enzymescantargetandsetinhighlyspecificreactionpaths.Italldependsontheenzymeand thephysiologicalconditionitfindsitselfin. EnzymesdonotacceleratealreadyGpositivereactions LectureEnd Calculatingproblems Question:CalculatetheEntropyorEnthalpyofareactiongiveninitialandfinalstates. Enthalpy:Initial5000(J*mol^1),Final12000(J*mol^1) Solution:H=H(final)H(initial),H=120005000 Answer:H=7000 Question:Isthereactionspontaneousornonspontaneous?H=7000,S=20,Temp=100 Celsius. Solution:G=HTS,G=7000T*20,TinKelvin=100Celsius+273=373 G=7000373*20 Answer:G=460,sincetheGibbsfreeenergyvalueisnegativethereactionisspontaneous (exergonic). Question:WhatistheKeqequilibriumconstantofareaction?Standardfreeenergychange A>Bis20KJ*mol^1,T=300Kelvin Solution:Keq=e^(Gdegree/RT),Keq=0=e^(20000/(8.314*300)) Answer:e^(8.019)=3037 Question:Whatistheactualfreeenergychangeofareaction?A>Bat300Kelvin,A=1mol,B =.01mol,Gdegree(standardfreeenergychange)=20kJ Solution:G=Gdegree+RT*ln*([B]/[A]),G=20kJ+8.314*300*ln*(.01/1) Answer:G=31kJ

2ndLectureWednesdayAugust28th,2013Water ProductsoverReactants (C^c*D^d/A^a*B^b)intheG=Gdegree+RTln([C^c]*[D^d]/[A^a]*[B^b]) Waterfacts Watercovers70%ofearthsurface. Watermakesup70%ofvolumeofbody.

2ndmostcommonmoleculeinUniverseafterHydrogengas. Waterhasspecialproperties,thatareimportantforlifetoexist. Watermediatingstructureofchemicals Biochemicalmoleculesareverylarge.Theirstructureismediatedbywater.Surroundingwater determinesitsstructure. Wateractivelyparticipatesinreaction,especiallyitsionsH+andOH. Shapesandthereforefunctionsofbiologicalmoleculesaredeterminedbythechemicaland physicalpropertiesofwater Reactionsaremediatedbywater Structureofwater Lavoisiershowedwaterisnotanelement. Nonplanaroxygenhaslonepairsofelectronsassociatedwithit.Occupiessimilarspaceas thehydrogenatoms.Sp3hybridized.Thisformsatetrahedralarrangementstructurewiththe lonepairs.Nonplanarwaterbondangle(104.5degree). Oxygenpartialcharge=0.66e hydrogenpartialcharge=+0.33e =>Dipole AllbiochemicalreactionstakingplaceinthisDipoleenvironment. Purewater=55molar OxygenHydrogenatombond=1angstrom Angstrom=1*10^10meters Hydrogenbond Onesidenegativelyonesidepartiallypositivelycharged.Twosidesattracteachother.This attractionistheHydrogenbond. 20kJ/molbondingenergy,Significantlylessthancovalentbond.(Covalentbondstrength 340460KJ/mol) Eachmoleculeofwatercanform4hydrogenbonds.Twolonepairsofelectronsaroundthe oxygencanactasacceptorsofahydrogenatomeachandtwohydrogenatomsthatcaneach actasanindividualdonor. 1.8Angstromsbondlength.Thisbondlengthisalways0.5shorterthanforacorrespondingVan derWaalswouldbe,thisexplainsitshigherattractiveforce. Eachhydrogenbondissmall,butintotaltheoverallcontributionisverysignificant D=Donor,partiallypositivelycharged A=Acceptor,partiallynegativelycharged AnypolarmoleculecanformaHydrogenbond,ifithasalonepairofelectronsthatitcandonate.

Hydrogenbondsalwaysbreakingandreforming,every2*10^11seconds. Inwatereachmoleculeisundergoing4hydrogenbondsateachtime.Howevercomparedtoice, thesebondsaredistortedandirregularandthereforewaterisnotrigid.Eachbondbreaksupat theabovegivenspeed. Bondstrengthcomparison CovalentbetweenOxygenHydrogen340460kJ Ionic86kJ Hydrogen20kJ DipoleDipolebetweencarbonylgroups10kJ Londondispersion(vanderWaals)1kJ(electrostaticinteractionbetweenatoms) Bondlengthcomparison OxygenHydrogenBond HydrogenBond VanderWaals

1Angstrom 1.8Angstrom 2.3Angstromsor0.5morethanthecorrespondingHydrogenbond

ICE Wateristheonlysolidthatfloats.Icelessdensethanwater. Formarigidarrangementsoeachmoleculecanformmaxnumberofhydrogenbonds. 4bonds,allthehydrogenbonds,formtetrahedralshape. Freespacebetweenwatermolecules. Boilingpoint Waterhasextremelyhighboilingpointcomparedtoothermoleculeslikemethane. Duetohydrogenbonds,eachbondisbreakingevery2*10^11sec(picoseconds). Solvent Waterwilldissolvemorecompoundsthaneverythingweknow,bestsolventaround. Hydrophilic substancetendstodissolveinwater.Generallypolarorcharged. Hydrophobic substancesdonotdissolveinwater.Generallynonpolaroruncharged. Todissolve=Interactionbetweenionsandwaterhastobestrongerthaninteractionsofsalt ions. Watersofhydrationionsthatgetsolvated(surrounded)bywater. Saltsdissolving Saltbecomessolvated(surroundedbywater)andthereforehydratedifthewatersaltion hydrogenbondsarestrongerthanthebondsbetweenthecationandioninthesaltitself. Hydrophobiceffect HydrophobiceffectThephenomenonthathydrophobicsubstancesclingtogetherwhenplaced intoapolarsolventlikewater.Theydonotdissolve,butareexcludedfromwater.Nonpolar moleculesgetexcludedandwatersentropygetsmaximized.Nonpolarmoleculesstick

together. Experiment:Nonpolardissolvedinwaterstartsaggregatingtogetherduetohydrophobiceffect. TheGibbsfreeenergyforthisprocessisnegative,thereforespontaneous.TheHis endothermic(positiveH)orisothermic(0H),whichsuggeststheyareenthalpicallyspeaking nonfavorableorequal.SoSincewehaveG=HTS,theShastobepositive(entropically favored,moredisorder)andbig.Theprocessmustbeentropicallydriven. Entropydecreaseswhennonpolarmaterialenterswater.Meansitmustbemoreordered. Alotofthiscomesfromthewaywaterordersaroundthenonpolarmolecules. Waterusuallycancreate4hydrogenbondsinregularwater,sothereare4waysofmaking bonds.Thesebondsgetbrokenandreformedveryfast,andtherearealotofwaysofarranging (highentropy,ordisorder).Ifoilisfilledintowater,firstthingthathappensisoilstartstocling togethermoreandmore.Thewateratomshavetoarrangearoundtheoilbubble.Themolecules cannotcreatehydrogenbondswiththeoilbutonlywiththeotherwatermolecules.Thebreaking andformingofnewhydrogenbondsisimpededbecausetherearelesswaystocreatethe hydrogenbonds(noncanbemadewiththenearbyoil).Thisiswhyentropydecreases,orit becomesmoreordered).Sowhenoilstartstoformbiggerbubbles,waterhastodolessof thesestructuresaroundoil(lesssurface),waterbecomesmoredisordered. ThiscausesGtobepositive(becauseScomesnegative)andtheprocessisunfavorable.The surfaceareaoftheoiltowaterdecreasesbecausetheoilbubblesaggregatetogether,thereby maximizingtheirpossibleentropy. Inshort Waterformscagestructuresaroundhydrophobicmolecules.Bondsofwaterstaymuchmore rigid,switchesbondsmuchless,createsmuchmoreorderinganddecreasesentropy. Amphiphiles Havebothpolarandnonpolarparts,thereforebothhydrophobicandhydrophilic,dependingon whichpartyoulookat. Amphiphilicmoleculesinwaterhidehydrophobicpartsandexposehydrophilicparts. Twoforms:Micellesandbilayers Micelles Spherearrangementofamphiphiles.Hydrophobicpartinwards,hydrophilicpartcreatessurface whichinteractsthroughhydrogenbondswithwater. Bilayers Hydrophobictailhidden,hydrophilicheadoutward,justinabilayer.Thehydrophobictailsdont bondwitheachother,buttheyexcludethemselvesfromwaterduetoentropicreasonswater canformmorehydrogenbonds,higherentropy,ordisorder.Londondispersionforcestooweak toaccountforit.

ColligativeProperties Aphysicalpropertyofasolutionthatdependsonconcentrationofdissolvedsubstancesrather thanontheirchemicalproperties. Solutesdepressfreezingpoint,elevateboilingpoint Osmosis Movementofwaterfromlowsoluteconcentrationtohighsoluteconcentration OsmoticpressurePressurethatmustbeappliedtocounteractosmosisorstoposmosis. Dependsdirectlyonconcentrationofsolution. Always22.4atmfor1M Diffusingwaterrandomlydistributesitselfthroughawalltoequalizeosmoticpressure,oruntil concentrationsonbothsidesareequal.Thermodynamicallyfavored,duetoincreaseinentropy. Entropicallydriven. ChemicalpropertiesofWater WaterionizestoformH+andOH Inrealitynofreeprotonsrunningaround.TherearereallyjustH3O+ Protonsjumpfromwatermoleculetowatermoleculereallyquickly.OHdiffuseveryquicklytoo. ThisisthereasonAcidBasereactionsareamongthefastestwecanmeasure(duetoproton jumping) Dissociationconstant: K=([H+][OH])/([H2O]) Theconcentrationofunionizedwaterissolargewecanignoreit. Thereforeforwaterthedissociationconstantis: Kwater=[H+][OH] Onlysmallamountofwaterionizes(10^14).Equalamountofprotonsandhydroxideion. Purewaterthereis10^7molarH+andOHions,concentrationalwaysequal. Addextraprotonstherelationshipchanges.Butrelationshipstillreciprocal.Increaseprotons, concentrationofhydroxidegodown. pH=log[H+]=log(1/[H+]) pk=logK Waterisacidandbase WeuseBronstedandlowrydefinitionofacidandbase Acid: Substancethatcandonateaproton Base: Substancethatcanacceptaproton

HA+H2O=H3O++A A istheconjugatebaseoftheacidHA H3O+ istheconjugateacidofthebaseH20 Watercanfunctionasbothacidandbase,dependingonthesituation. Includingwatertheformulaforabasicsubstanceis: HB+=H++B Dissociationconstant Definesthestrengthofanacid. Reactantsonbottom(denominator),productsontop(numerator). K=([H30+][A])/([HA][H2O]) Waterisessentiallyconstantat55.5M.Itcanbedeletedoutoftheequationandweusetheterm Katodescribeit. Ka=K[H2O]=([H30+][A])/([HA]) UsingpKsinsteadofpH SometimesweneedtousetheKvalue(disassociationconstant). pK=logK Withthisformulaacidsareclassifiedastohoweasilytheytransferprotonstowater. K<1 Isaweakacid.Onlypartiallyionizeinaqueoussolutions. K>>1 Isastrongacid.Transferalltheirprotonsintowater. ThestrongestacidisH3O+andthestrongestbaseOH pHofasolution HendersonHasselbachequation. pH=pK+log([A]/[HA]) Howevercannotaccountforionizationofwateritself.Andisnotgoodforstrongacidsorbases. Buffers KeeppHsrelativelylevel.Bufferstypicallyareweakacids.Importantinbiology. Ifyouaddacidorbase,theywillequalizeit,orisinsensitivetofluctuations.Bufferingcapacity bestaroundpK

Titration CenteroftitrationcurveiswherepH=pK,ortheacidisequalinconcentrationtoits correspondingbase,[HA]=[A].Whereconjugateacid,baseisequalinconcentration. Polyproticacids Canloseseveralprotons.HavemultiplepKs,oneforeachionizationstep.Discretevaluefor eachstep. Ex.:H3PO4 Bufferingcapacityhowwellanacidbuffers.HitsitsmaximumwhenpHclosetopKorwithin onepHunit.OtherwisethepHchangestoorapidly. TomakethebuffersolutionatacertainpH,wechoseabufferwithbufferingcapacityaroundthat pHandaddequalamountsofacidanditsconjugatebase. CalculatingProblems Question:10^5molofHClisaddedto1literpurewater.WhatisthepHafter? Solution:PurewaterhaspH7=10^7molofH+.Theacidhas10^5molH+.ThereforetotalH+ is10^5. Answer:pH=log[H+]=log(1.0*10^5)=pH5 Question:WhatisthepHofa1Lsolutioncontaining20mlof5Maceticacid(pk4.76)and 10mlof2Msodiumacetate? Solution:(20ml*5M)/1000mL=.1Maceticacid,(10ml*2M)/1000mL=.02Msodiumacetate. pH=4.76+log(.02/.1)=4.760.7 Answer:4.06 Question:A1Lsolutioncontains.2Mformicacid(pK3.75)and.2Msodiumformate.Weadd2 mlof1MNaOH.CalculatethepHbeforeandaftertheadditionoftheNaOH. Solution:before:pH=pK+log(A/HA)=3.75+log1/1=3.75+0=3.75 after:2ml*1M/1002ml=2mM.SincetheNaOhwillneutralizetheformicacid.Sotheformicacid goesdownby2mMandtheconcentrationofsodiumformategoesupby2mM. pH=3.75+log([.202]/[.198])=3.750.008 Answer:3.742

Lecture3FridayAugust30thAminoAcids StructureofAminoacids Rgroupcanbehugeorganicsidechain 20differentnaturallyoccurringaminoacidscanbesidegroups(19amino acid,1iminoacid)

Aminoacidsaredipoles.AminehasahighpK,CarboxylgrouplowpK Inorganisms,waterorpH7carboxylgroupindeprotonatedandaminegroupinprotonatedstate. MoleculehasplusandnegativeioncalledZwitterion pKisthepHatwhicheachformofacidandconjugatebaseisatequalconcentration. Peptidebonds Aminoacidsundergocondensationreactionstocreatepolypeptides,aminoacidslinkedby peptidebonds.Aminoacidsinpolypeptidesarecalledresidues. Peptidebondcreationcalledcondensationreaction,waterisformedasproduct. Chargeonthecarboxylateandaminearelost,exceptforontheNandCterminus. Primarystructure SequenceofaminoacidsfromNCterminus Aminoacidsidechains Memoriseaminoacidsandtheircharacteristics. Sideofsidechainshavebigroletoplay.Chainscanclashwitheachother. Nonpolaraminoacids Alanine,Isoleucine,Phenylalanine Sidechainscanclashwitheachother,especiallybig phenylalanineones.Nonpolarsidechainsclingtogetherwhenin water.Hydrophobicpartgetshiddenwhilehydrophilicpartis directedtowardwater. Serine(RCH2OH),Guanine Twoserinebondscanformhydrogenbondsbetweeneachother, orwithotherpolarunchargedaminoacids.

Unchargedpolar

Cysteine(unchargedpolar) Cysteinehasthiolgroups(RSH)whichcanformdisulfide covalentbondswitheachotherthroughcondensationreaction. Disulfidebondsonlyforminoxidizingenvironment,insidecells howeverareusuallyreducingenvironments.Disulfidebonds dominateinproteinsthatareexcretedtooutsideofcell(blood proteins,extracellularmatrixproteins,etc.) Chargedpolar Aspartate(RCOOH),Lysine(RNH2) Tendtocoversurfaceofprotein,wheretheycaninteractwith watermoleculesinaqueousenvironment.

Aminoacidnames(memorize)

Glycine Gly Alanine Ala Valine Val Leucine Leu Isoleucine Ile Methionine Met Proline Pro Phenylalanine Phe Tryptophan Trp Serine Threonine Asparagine Glutamine Tyrosine Cysteine Lysine Arginine Histidine Asparticacid Glutamicacid Ser Thr Asn Glu Tyr Cys Lys Arg His Asp Glu

G A V L I M P F W S T N Q Y C K R H D E

Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar(iminoacid) Nonpolar Nonpolar Unchargedpolar Unchargedpolar Unchargedpolar Unchargedpolar Unchargedpolar Unchargedpolar Chargedpolar Chargedpolar Chargedpolar Chargedpolar Chargedpolar

Specialcase Asx Specialcase Glx

EitherAsnorAsp.Reason:Onecanhydrolyseintotheother. EitherGluorGln.Reason:Onecanhydrolyseintotheother.

Multipleionizablegroups Ifanaminoacidhasmultipleionizablegroups,theirindividualpKvaluesdependonthenearby groups.Canonlybeexperimentallydetermined. BecauseifyouhaveaminesidechainnexttonegativelychargedgroupitspKwillbedifferent. AtlowpHs(ex.pH2)thehighconc.ofprotonswillmakethecarboxylicacidRCOOHandthe aminegroupRNH3+beprotonated.AthighpHs(ex.pH10)thehighconc.ofhydroxylgroups willmakebothbeunprotonated. Isoelectricpoint pKofentireprotein.pHatwhichthereisnonetchargeonanaminoacid.Alwaysneedtobe experimentallydetermined,cannotrelyoncalculations,becauseinrealityvaluesdifferfromwhat theyarelisted.Pointatwhichthereisnonetelectriccharge,notatwhicheverythingis uncharged.Positiveandnegativechargescanbeequal. pI=(pki+pkj) Nomenclature

needtofamiliarizewith3lettercode,andsinglelettercodeforexam. StartfromNTerminusandgotoCTerminus. Regular AlanineTyrosineAspartateGlycine Threeletter AlaTyrAspGly Oneletter AYDG Chirality AminoacidshavechiralcentersatCalphaCarbons(4attachmentshavetobedifferentfrom eachother).LandDaremirrorimagesofeachother,enantiomers.Allweneed,wedontneed RandSsystemlikechemists.Laminoacidsnotnecessarilybendlighttoleftinpolarimeter, andneitherdoDaminoacids.DandLonlyrelevantforaminoacidconfiguration. WewilluseFischerprojection.Doesnotshow3Darrangement.Forthatweneedtoknow,that: Ifbondisdrawnvertically,thenitsgoingintobackofplaneinreality.Ifthebondisdrawntothe side,thenitwillgointothefrontofthepaper.Noneedforwedgesanddashes. OnlyLaminoacidsfoundinbiologicalsystems. Chemicalsynthesis racemicmixturesofbothenantiomersmade Biosynthesis onlyLaminoacidsaremade.Becauseplatformthatismaking moleculesislefthandedtoo. Sidechainmodification Nonstandardaminoacidsarecreatedafteraminoacidsarecreated.Modificationshappenvia enzymes. Example:OPhosphoserine.Serineattackedbyphosphategroup andphosphorylated.Phosphorylationofserineturnsmanyproteins onandoff.Phosphorylationalsoimportantinsignalingmolecules. Example2:GABAanddopamine.Modifiedforneurotransmitteruse.Histamine,modifiedfrom histidinebydecarboxylation. Example3:GFP.ModificationisspontaneouswithSerTyrGlysequence.Asitfolds,itfolds itselfintoGFPspontaneously.Dependingonenvironmentitcanbedifferentcolor.Differenceis onlyslightchangesinchemicalenvironment. Canusethisphenomenon.ByfusingthesemoleculestogethertoGFPtolocatewhereproteins areincell. EndofLecture

CalculatingProblems Question:Calculatetheisoelectricpointofglutamicacid.pk1(alphaCOOH)2.10,pk2

(alphaNH3+)9.47,pkR(gammaCOOH)4.07. Solution:TheformulafortheisoelectricpointispI=(pki+pkj)/2.Whichpkstousethough?In thiscasewewillusethepk1ofthealphaCOOHandthepkRoftheRGroupCOOH.Thebook offersnorealexplanation.Onecanassume,thatbecauseitisanacidyouwouldwantto neutralizetheacidparts,becausewearelookingfortheisoelectricpoint,orpointwherethereis nochargeonthemolecule. Answer:pI=(2.1+4.07)/2=3.085

Lecture4WednesdaySeptember4thProteinpurification Proteinpurification1stthingtodoinchemistry Everyproteinisdifferent,hasdifferentproperties.Weneedtofindwaystoseparateproteinson thebasisofthesecharacteristics.Moreimportanttomakeproteinpurethantominimizelossof protein. PrimarystructureAminoacidsequenceofpolypeptidechain.Propertiesofproteinare affectedbyprimarystructure.Nottalkingabout3Dstructure,onlylinearchain. Limitingfactorsofprimarystructure: <40residues hardtobuildstapleshapeandcarryoutfunction 100residue/aminoacidlimit difficulttofold 1000residuelimit difficultywithmRNAproducingit,makemoreerrors. Verylargepolypeptidechainsusuallynotonesinglechain,butseveralputtogether.Givesusthe abilitytobuildbiggerconstructs.Theoreticalboundaryislimitless. Mostaminoacidshave1001000residues. Ifgeneralpolypeptidescalledproteinswhenmorethan40residues,belowthattheyarecalled peptides. Cells Notabagofwater.Verycrowdedenvironmentwithmanydifferentkindofproteins.Tostudy themweneedtoseparateproteins. Purification Easiestwaytopurifyaproteinistohavealotofitinthefirstplace Conditions Conditionsproteinsarehappyinarecontrolledenvironments pH OnlystableinnarrowrangesofpH.HighorlowpHcanaltertheprotein,usually useabuffertostabilize. Temperature Candestabilizeproteins(mesophilicorganisms,onesthatarehappyat37 celsius).Theydenatureproteinsathightemp.Proteinsneedtobeatlow

Enzymes

temperature. Arounddegradativeenzymescanharmit.Theseenzymescanbeinhibitedby addingmoleculesthatblockthemorchangingpHsotheyarentaseffective.

Spectroscopy Quantifyhowmuchproteinwegot. Usesdifferentlightabsorbancepropertiesofchemicals Proteinstakeinlight,wecanseehowmuchitabsorbs BeerLambertlaw: A=log(l0/l)=e*c*L A=absorbance(unitless) e=extinctioncoefficient,orabsorptivity c=concentration L=pathlengthoflightgoingthroughsample I0=Lightgoingoutofsolution I=Lightgoingintosolution Proteinswillabsorblightatdefinedwavelengths. UseUVlight,aromaticringsabsorbthislightatspecificUVnmranges.Aromaticringamino acidslikeTry,Trp,PheeasytoseeinspectroscopybecausetheycanpickupUVlight.Absorb lightatspecificwavelengths.Soifyoutryingtoquantifyaprotein,useonethathastryptophanor anyotheraromaticring.Makesiteasiertosaywhichproteinsitis. PurificationSaltingout Usuallyusedasfirststepinpurification. Saltingout(alsocalled:ammoniumsulfatecut,highsulfatecut). Itseparatesproteinsonbasisoftheirsolubility. Procedure: Addhighconc.ofsalttomaketheproteinsmoresoluble.Asyouincreaseconcentration,salt ionscompetewithwatermolecules.Sothewatermoleculesgowayfromproteintosaltions,its hydrationshellgoesawayandtheproteinsdropoutofsolution. Dependingonstructuretheseproteinsdropoutatdifferentconcentration.Goodwaytogetridof contaminants.Proteinsprecipitateoutatdifferentsaltconcentrations. Goodforinitialstep,notgoodforveryfinepurification(cangetridof50%ofcontaminantsaway). ChromatographyGeneral Mobilephase(liquidorgas)containsourmolecule.Stationaryphaseiswhereitson(paper, ceramicplate).Mobilephasewillinteractwithstationaryphasetodifferentdegrees,depending ontheproteinpropertyanditsinteraction. Canusedifferentproteinpropertiestoyouradvantage:Charge,polarity,size,bindingspecificity.

ChromatographyIonexchange Anionexchange Negativelychargedproteinsbindtosolidcationicmatrix(stationary phase) Cationexchange Positivelychargedproteinsbindtosolidanionicmatrix Proteinsareusuallynegativelycharged,rarertofindpositivelychargedproteins. Alterconditions PH Salt

Makeproteinsmoreorlesscharged,toselectconditionsatwhichto separateproteins. Cancompetewithproteinforbindingtomatrix.

Procedure: 1)Columncontainsstationaryphase.Samplemixturegoesthroughmatrixwithlowsalt.If proteinisnotchargedatallitwillfloattillbottom(bestsolution).Mostofthetimeproteinswill interactlessormorestronglywithit.Addmorebuffertoseparateproteins.Thebufferwillwash proteinsdownwards.Theproteinsthatstickthebestwillgodownwardtheleast.Thiswayyou canstartseparatingthem.Thenaddhighsalttodistinguishproteinsmore.Gradientof increasingconc.ofsalttoseparateproteins.Dependingonconcentrationproteinswillgetsolved outatbottom. MonitorprogressandwhichproteinsaresolvedoutatbottombyUVlighttoseewhichproteins areextracted. Wanttobeabletoquantifyandseparateproteins.Graphwillshowdifferentpeaksfordifferent proteins. ChromatographyHydrophobicinteraction Separatesbynonpolarity.Increasinglengthoftailsincreasestrengthofhydrophobicinteraction. Theamountofhydrophobicinteractionsareatsurfaceareexploitedinthechromatography.Do notstartatlowsaltbufferlikeinIonExchange,butinhighsaltbuffer,becausehydrophobictails willinteractmoreweakly. ChromatographyGelfiltration Separatesmoleculesbysize.Nointeractionusedatall.Sortingproteinsdependingonsize. Beadswithporesincolumn.Someproteinscanenterporesandotherscant.Smallproteins canenter,exitporesandhavetotakesignificantlylongerpaththroughthebeadscomparedto largeproteinswhichjustgoaroundbeads.Largeproteinscomeoutmorequickly. Largestproteinswillnotenterporesjustflowsonoutsideofglass.Thisiscalledvoidvolume. ChromatographyAffinity Exploitsthespecificbindingbyproteins.Someproteinsbindtospecificmoleculesthatarefixed inourchromatographycolumn.Specificproteinswillbindtomoleculeinmatrixwhileothers

wont.Canveryspecificallytargetproteins.Highestpowerofallchromatographymethods. Havetoknowwhichproteinyouarelookingfor. Ifyoudonotknowwhichproteinyouarelookingforyoucanuseanimmobilizedmetal.Addtag toendofprotein,likeHistidine.Histidinebindstonickel.SoeverythingthatHisTagwillstayin column. Electrophoresis Separatesmoleculesaccordingtochargeandsize calledPAGE:Polyacrylamidegelelectrophoresis DiffersfromGelChromatographyinthatsmallermoleculespassfaster. Putitintogelwhichhaspores.Putelectriccurrent,whateverhasmorenegativechargegoes downmore. CoatproteinswithSDSandnowtheywillallmoveintonegativecurrent.Willmaskanycharges. Willmakeproteinsmoveaccordingtomassnottocharge.(separateacouplehundred,difficult toseparatemore),whileregularPAGEwilldoboth. Twodimensionalelectrophoresis(separate1000sofproteins).Importantinproteomics. Thistimewedoseparatebasedonchargeaswell. 1)Firstexperimentseparateoncharge(isolectricfocusing). Tophasgradientofphfromlowtolefttoright.Eachproteinwillmovetoisoelectricpoint, becausethisiswhereitwillhavenomorechargeandnotbeattractedtomovefurther.Itwould movebacktoisoelectricpointifitmovestoofarpastit. 2)SecondpartisSDSPAGEwhereweseparateonsize.Thisisdonedownward. Doneinproteomicexperiments,toseeallproteinsincell. Ultracentrifugation Assay Proteinscanbequantifiedbyassays.Inorderword,ittellsyouwhichproteinyouhave. Locksforactivityofprotein.Usecatalysisenzymebecausetheyhavespecificsubstratesthey bindtoandarereadilydetectable.Measurethatcatalyticreaction,thatactivityisrelatedtothe amount. Procedure: 1)Haveantibodiesonsolidfoundation. 2)Havetheproteinyouarelookingtomeasurebindtotheantibody 3)Havesecond(freefloating)antibody,bodythatislinkedtoenzymebindtoprotein. 4)Enzymecatalyzesreaction.Dependingonhowmanyreactionstakeplace,youknowhow manyproteinsbound. Ifthereisnobioactivity,youknowyourproteinisnotthere.

Lecture5FridaySeptember6thBiochemProteinSequencing Proteinsequencing Allstepsarebasicstepsneeded,nomatterifprocessisoldornew.Eveninmodernprocesses thelargepolypeptidechainsneedtobebrokenuptobesequenced.Even40100polypeptide longsequencescannotberead. 1)Determinedifferenttypesofsubunitpolypeptidechainsareinproteinlinkedtogether.How manyarelinkedbydisulfidebridges Reaction: Usedansylchloridewhichwillreactwith3aminochainlength. 1)ReactswithNTerminalaminogrouptoformadduct.Doneunderbasicconditions. 2)Adductchaincanbeexposedtoacid.Acidwillhydrolyzeallpeptidebonds.Dansyl chlorideisfluorescentyellowandcannowusechromatographytogetitout. ThiswaywecandeterminewhataminoacidofNterminalis.Andwecanseehowmany differentNterminithereare,iftherearemultiplepolypeptidechainsthereare. 2)Nowafterdetermininghowmanypolypeptidesthereare,disulfidebondsmustbecleavedto separatesubunitpolypeptidechains. Reaction: 1)Exposedisulfidebondstoreducingagents(2Mercaptoethanol).Redoxreaction, wheredisulfidebondswillbereducedtotwothiolgroups. 2)Wanttoblockcysteinedisulfidebondreformingwithiodoacetate.Makes carboxymethylcysteine,itcannotgoback,isanirreversiblereaction.Cysteinenow blocked. 3)NowweknowhowmanychainsthereareANDwehaveseparatedthem.Nowweneedto fragmentthemtothepointwheretheycanbesequenced.Means:lessthan30aminoacidlong chains. Twowaysofdoingitenzymatically,orchemically(chanceofdying). Reaction(enzymatically): 1)UseProteaseswhichcleavelargepolypeptidestoproducesmallerfragments.Wewill useTrypsin(Try).DifferentProteaseswillcleaveproteinsatdifferentsequences. Placeitlikestocutiscalledspecificity. Trypsinspecificity:Rn1=positivelychargedresiduesArg,LysRnun=Pro.Meansthat residuetowardsNterminushastobeArgorLysandtheresidueafterthat(formingthe peptidebondwithit)cannotbePro. Reaction(chemically):

1)Cyanogenbromide.Modifiesonlymethioninesulfur.Doesnottouchcysteinesulfur. Createscovalentbondwithsidechains.CyanizationofaminoacidatNTerminusof peptidebond.BreaksofftoCH3SCNandtheNterminusofthepeptidechainendsup formingPeptidylhomoserinelactone.CTerminuspartofchainwillhaveitsaminegroup deprotonated(itwillbepositivelycharged),freeNTerminusonCchain. Cyanogenbromideinwaterwillhydrogencyanideacidwhichistoxic. 4)SequencingreactionEdmandegradation(pre1995) Reaction: 1)Reactingmolecule(PITC)formsanadductwiththefirstaminoacidatNTerminus. Worksbetterthandansylchloride. 2)AnhydrousTFA(Trifluoroaceticacid)willcleavethepeptidebondwiththismodified NTerminusaminoacid.OnlycleaveNterminal1aminoacidpeptidebondwithsecond aminoacid). 3)Nowwehaveoriginalpeptidechainminusonepeptideandthiazolinonederivative.Add acidandthisbecomesPTHaminoacid.Itisinaqueousenvironmentsowecan separateitbecauseitisveryhydrophobic. 4)Thisactionisrepeated.AlwaysdeterminetheidentityofnextNTerminal.Originally donebyhand,nowitsautomated. LimitationsarethatyouneedfreeNTerminus.Notallarefree.Inhumans50%ofNTerminiare acetylatedormodified.Ifyouhaveitmodifiedyoucannotdothisverywell.Thatiswhyitonly worksverywellwithbacteriageneswhicharentasmodified. 3)CanusemassspectrometryifNTerminusismodified.Massspectrometryseparates moleculesdependingonmassandcharge.Ionizesthemandmagneticfieldwilldeflect moleculesdependingonmassandcharge. ElectrosprayIonizationmassspectrometryovercomesphenomenonofbigmoleculestofall apartwhenionizedbymassspectrometry. Reaction: Solutiongetsshotout. Nitrogengasfiltersoutsolvent. ESIisaccuratewithin0.01% Tandemmassspectrometryforpeptidesequencing(REDO) 1)Ionizedpeptidesfromproteasedigest Blastittoafilter,oneionizedpeptidegoesthrough. 2)Goesintoheliumchamberwhereitfragmentsagain. 3)Analyzefragmentsindetector. Doitmultipletimestoanalyzeeverysinglefirstfragment.Putitalltogetherinend.

Proteindatabases Collectionofknownproteinsequences. Usingproteinsequenceinformation Aminoacidsequencesareverysimilartoanimalsthatareclosefromanevolutionary perspective. 1)Homologousproteinsarerelated.Geneduplicationmakestwocopiesofgene.Onegenecan keepdoingitsoriginalfunction,othercopycanevolvetodoadifferentfunction. 2)Differentproteinsevolveatdifferentrates.Dependsonhowlikelyitwillbethatthatmutation willbeviableandtolerated. 3)Manyproteinsareconstructedfromdomain.Domainsareunitsoffunctionality,orself containedfoldedunit.Usually100200aminoacidsmakeadefinablestructure.Thesedomains popoverandoveragainindifferentproteins,evenifunrelated.

Lecture6MondaySeptember9thSecondaryStructure Secondary Tertiary Quaternary 3Dsubstructure,includingalphahelixandbetasheets. secondaryfoldsuponitselfintosubunits. onlyappliestoproteinswithmultiplesubunits.

Peptidebondconformation Peptidebondisplanar,canadoptoneoftwoconfirmation(cisandtrans).40%doublebond character.Westillgetsomerotationbetweenplanarpeptidegroups. Transconformationismostcommon.Aminoacidsareonadjacent sides. Cisconformation8kJ/mollessstable.Aminoacidsareonsame side.Usuallyexistinprolineresidues(10%ofprolineresidues) Rotations Polypeptideisstringofplanargroup.Norotationwithinpeptidebond. ExtendedconformationhavedifferencesbetweenCalphasof2.5angstroms. Rotationsarebetweenplanesofgroups,meansrotationbetweenCandOorN andnotinpeptidebond. Phiangle alphacarbonandnitrogen Psyangle alphacarbonandoxygen Intheorycanhavefreerotation.Inrealitycertainrotationsbetweenphyandpsy yougetsterichindrancesbetweensidechainsandaminoacids. Thatiswhyinpracticeonlysmallamountofpossibleanglesofwherepeptidebondscanform

doworm. DisplayedinRamachandranPlot RamachandranPlot White Forbiddenspace.Anglescausestericclashes. Black/blue Favorableones Green Toleratedbutnotpreferred Exceptions Proline Glycine

Allconstrainedbecauseofsidechain. Doesnothaveasidechain,doesnothave forbiddenspace.

Recap: Twothingstothinkaboutwhichrotationisallowedisplanarityofpeptidebondandclashes betweensidechains. Alphahelix Mostcommonandrighthanded. 3.6aminoacidsperturnanda5.4Angstrompitch(heightatwhichitextendsupwards) HydrogenbondsbetweenC=OofNthresidueandNHofNth+4residue. Betasheet Aswithalphahelix,itfulfilsallofitspossiblehydrogenbonds. Hydrogenbondsbetweendifferentchains,canbebetween2differentregionsofsame polypeptide.Twotypesoforganization. Antiparallel Strandsruninoppositeofthemselves(leavesperfecthydrogenpattern). C>NandN>Cinoppositedirection.Basicallychainmakesaloopand thenalignsitselfwiththestrandinoppositedirection. Strandsruninsamedirection,arrangementofoxygenbackboneleaves distortion(lessstable).Chainloopneedstocrossovertoformparallel strandnexttooriginalstrand.

Parallel

Sheetshavepattern.Usuallybetween2and22strands,usually567strands.Parallelusually needmorestrands. Sidechainsalternateaboveandbelowtheplaneofthesheet.Sidechainsalternateaboveand belowofthesheet(up,down,up,down,etc.) Betabends TypeI TypeII

Loopsandturns. Regionsbetweenalphahelicesandbetasheetsarecalledloopsandturns.Sometimesorderup toformsecondarystructure.Usuallyonoutsideofprotein.Alphaandbetasheetsareusuallyat coreofprotein Proteinstructure Proteinsareeitherglobularorfibrous. Keratin(fibrous) Twoalphakeratinhelicestwistedaroundeachothertoformahelicalcoiledcoil. 5.1angstrompitchinsteadofregular5.4inalphahelix Reason:Twoalphaheliceswraparoundeachothertoformcoiledcoil.Theseproteinshavevery definedprimarystructureandwhichmakeverydefinedsecondarystructure(alphahelix). Primarystructurerepeatsevery7residues.Thishappensonbothchains.Gethydrophobic residuestriponbothsidesofeachoneofthetwoproteinsonpositionsAandD.Repeatsevery 7residuetogetallhydrophobicmoleculesonAandD.Thispackagingstriprunsontheentire moleculeandpacksthetwostrandstogetherverytightly(dotoentropy). Coilingoftwohelicesisduetodifferencebetweentiltingofhelices.Slightdiscrepancybetween 3.5primaryresiduerepeatand3.6residuerepeatofsecondarystructure(???) HaveheadgroupsatCandNterminus,thatinteractwitheachothertointertwinefilamentswith eachothertoformprotofilament.4Protofilamentscometogethertoformmicrofiber. microfilamentscometogethertomakemacrofilaments,andmanymacrosmakehair. Alotofcysteinebondsinkeratinthatformdisulfidebonds.Dependingonhowmanydisulfide bondsyouwilleithergetstrengthinhairorstrengthinfingernail Increasingthenumberofresiduesperturnto4.1changespositionofhydrophobicaminoacids. Nomorelininguponsamesideofalphahelix.Changepatterning. Reduceyourenvironmentwillreducethedisulfidebonds.Disulfidebondsneedoxidative environmenttoform. Collagen Foundinbones,teethandconnectivetissue.Lefthandedindividualhelices,righthanded supercoilconsistingofthreeindividualhelices. Collagenisatriplehelixcontainingnonstandardaminoacids Thehelicesarentalphahelices. 30percentofresiduesareglycine,every3rdresidueisglycine.Restofaminoacidsareeither proline(withaddedhydroxylgroup)orsomeothermodifiedaminoacid. Formationofaminoacidsiscatalyzedbyenzymewithcofactorascorbicacid(vitaminC)

ScerviesnovitaminCandconnectivetissuecollagencannotbebuilt Primarystructure Isalwaysatripeptiderepeat.GlyXYwhereXisusuallyprolineandYisHyp/Hyl Prolinepreventsalphahelixfromforming. Formslefthandedhelixwith3aminoacidsperturn.Allformhydrogenbondswitheachother. Glycineisneededatevery3rdresidue,becauseelsethesidechainswontfit.

Lecture7 Collagencrosslinking Nocysteinetomakedisulfidebonds.Butcrosslinkedbetweenmodifiedlysinederivatives, Allysine.Upto4lysinescanbecovalentlybondedtoeachother. Reaction: 1)Lysinemodifiedbylysyloxidasetoallysine 2)Twoallysinefusetogethertocreatecrosslinkallysinealdol Thisallysinealdolcrosslinkcanbefurtherlinkedusingahistidinetocrosslinkupto4lysines together. Reaction: 1)AllysinealdolandhistidineformAldolHis. 2)Notherlysinecanbecomeattached Upto4heliceswillbeboundbythesecrosslinks. Secondarystructure Wecanpredictwhatsecondarystructurewillbejustbylookingatprimarystructure.Thereare ruleswhichaminoacidsliketobeinwhatsecondarystructure.Secondarystructureisthe foldingofthebackboneofthechain. Proline Helixbreaker.Orientationofsidechainpreventitfromperforminghelix. Iminoacidandcannotbepartofhydrogenbond. aHelix Rarelycontainsuccessivebranchedsidechains.Therewontbetwo branchedaminoacidsonsameside,becausenotenoughspacetofit themin. aHelixcapped CappedbyAsnorGlnbecausetheycanbendbackandformhydrogen bondswithhelix. Tertiarystructure Takesecondarystructureinteractionstofoldtheminonitselftofromtheactualsubunit. Arrangementofatomsin3Dspace.

Oftencaninferfunctionofproteinbylookingatstructure,especiallyforenzymes. Twomethodstoprovideatomicdetailofproteins,orarrangementofatoms. XRayCrystallography 1)HavepurifiedproteininSDSpage. 2)Growcrystalsfromthis.Addhighconc.ofsaltormoleculetocompeteawaywatertomakeit lesssolubleandhaveitsaltedout.Unlikesaltingouthowever,wherewejustaddtonsofsalt,we makeconditionsjustright,driveoutproteinslowlytheproteinwillformthisbeautifullooking crystal.Donotknowwhatthisamountofsalt/timewillbe.Gottotestitexperimentally. 3)UsehighintensityXRaybeamandshootitatcrystal.Crystalwillinteractwithbeamtoreveal itsstructure.XRayswilldiffractandwecanmeasurethepatternofdiffraction. 4)Makediffractionpatternforeverysinglerotationofproteintoget3Dmodelofit. Typeoflightwavetouse Usevisiblelight,thewavelengthistoolong.Cannotseethecrystal XRayshavewavelengthof1Angstrom.Sowecanvisualizethebondlengthsof2.5or2 angstroms.XRaysalsoarediffractedbyelectrons,sowecanlookatthedistributionof electronsintheproteinaswell.Wecanmakeelectrondensitymap. Nowwecanlookateverylittlepartandfigureoutwhattypeofmoleculesitis. Higherresolutionisimportanttousetogetitaccurate.Ifyoucanresolvetwopoints1angstrom awayyoucansayyourresolutionis1angstrom. 6Angstrommodelswehaveinitialconfidence. Majordisadvantageisthatwecanonlyseestaticrepresentation.Butinproteinsnothingisstatic, theymove,sowecantseehowitmoves. NuclearMagneticResonance NMRNuclearmagneticresonance Takesadvantageofthefactthatprotonsinatomicnucleiactasmagnets.Theywillresonateif putinmagneticfield.Howmuchtheresonatewilldependonthechemicalenvironmenttheyare in. Insmallmoleculesyoucandeterminestructureofwholemolecule,inlargeronesyoucanlook atlargerstructure (MISSING) NOESYexperimentdeterminewhichaminoacidsareclosetoeachotherin3Dstructure,evenif theyarenotlocatednexttoeachotheronprimarystructure. NMRproducesassembleofmodels,notasinglemodel. Overcomecrystallographydisadvantageofproteinsbeingstatic.NMRdoneinsolutionsoyou canseedistortionsandmovementofproteinsubstructures.

Canseedynamicinteractionsofbranches. Patterns Hydrophilicsidechainsarelocatedonoutsideofproteinandhydrophobiconinside,regardless ofprimaryorsecondarystructure. Motifs Motifsarrangementsofsecondarystructureelementsin3Dspace BaB Bhairpin aamotif FoundinDNAbindingproteins Greekkey Proteinclassification Doitinatertiarystructureway Canclassifyifmoredominantlya,Bormixofalphaandbetastructures Domain Proteinwithmorethan250aminoacidprobablywontfoldintosingleunitbutintomultipleunits calleddomains. Domainslargeunitsofpolypeptides Helpfulinanalysingthefunctionoftheprotein Domainsarefundamentalunitsofproteinevolution.Inevolutiondontreallychangetheir structurethatmuch,moretheirorientation.Soprimarystructurechanges,buttertiarystructure notthatmuch.Structureisconservedmorethansequence,evenonesthathavelongdiverged inevolution.Domainsconservedevenifputindifferenttypesofprotein,canbetunedtodo specificbiologicalfunctions. Proteindatabank Everyproteineverydeterminedisinit.EitherbycrystallographyorNMR. ProteinsareclassifiedaccordingtoitsstructureCATH Class>Architecture>Topology>Homology Wecandothisbecausetheyhaveveryconserveddomains. Class Eitheralpha,beta,bothornosecondarystructure Architect Grossarrangementofsecondarystructures Topology Whatisitlike,isitclosetoex.hemoglobinetc.Connectivityofstructures Homology Showsitasamemberofagroupwithacommonancestor. Andwegodownhierarchy. Quaternarystructure Canonlymakepolypeptidesuptocertainsize.Needmultiplesubunitproteins.

Multiplesubunitproteinscalledoligomers Eachindividualsubunitiscalledprotomers Interfacesarethespacesbetweensubunits.Thisallowsforcommunicationbetweensubunits. Moreoftenthannotoligomershavesymmetries. Capsidsarethebiggestoneswith180protomers.

Lecture8Proteinfolding Proteinsfoldmuchquickerthanweexpectthemto. Proteinstability Mostimportantinteractionforstabilityisthehydrophobiceffect.Itdrivesthemtofold.Ifyou calculatefreeenergythefoldedproteinisonly0.4kJmolmorestableperresidue(reference hydrogenbondis20kJ).Sostabilitynotstrong,buttheoneitdoeshaveisfromhydrophobic effect. Hydropathy Theamounttheaminoacidcontributestohydrophobiceffect. Theyhigherthehydropathythemorelikelytheywillbeoninsideoftertiarystructure. Fromgraph,ifwehaveastretchthatishydrophobicthenitprobisininteriorofmolecule Otherbonds,likehydrogenbonds,theydontaddtostabilitytoomuch. Hydrogenbondandelectrostaticforcesonlyimportantfordefiningnatureoftertiarystructure,not stability. Reason:Thehydrogenbondisjustaslikelytoformbetweensidechainsofaminoacidsand withwater.Doesntmatterifitsmakingbondswithatomsofmoleculeorwaterswimmingon outside. Metalions Stabilizesmalldomains. Domainsonlycoupleof100molecules.Ifaregionisshorterthanthatitcanbeunstable.Metal ionsstabilizethem,ex.Zincfingermotif. Zincfingermotif Locksin4sidechains. Coordinateswithalotofaminoacids(cysteines,histidines,aspartates,glutamates).Canhave widerangeofprimarystructures. Singleoxidationstate.Itwillneverbeinvolvedincatalysisbymovingfromoxidationstateto oxidationstate. VeryoftenfoundinDNAbindingproteins,liketranscriptionfactors.

Denaturing/DestabilizeProtein Heat Moleculesshake,untilinteractionfallsapart.Usecirculardichroism. Measuresamountofsecondarystructureinprotein,cooperativeunfolding ofstructureastemperatureincreases.Findpointatwhich50%ofprotein unfolds. Detergents Sodiumdodecylsulfate(SDSpage),disturbsinteractions,shields hydrophobicgroupsandallowsthemtounfold. Chaotropicagents Guanidiniumion,urea,smallmoleculesthatallowthemtobehappierin aqueousmolecules.Reduceentropybydisturbingwatergridaround molecule.Increasesolubilityofnonpolarsidechains.Disrupthydrophobic interactions,buthowisnotwellunderstood. pH Enzymeshaveverysmallrangesunderwhichtheywillbehappy.Other pHwillmakethemloseorgainhydrogenandchangestructure. Experiment(1950)laidgroundworkforwhatweknowaboutproteinfolding. HaveRNAseA,smallproteinwithintramoleculardisulfidebond. Usechaotropetodenatureprotein,unfolding. Addreducingmercaptoethanoltocleavedisulfidebondsbacktothiols. Result:Denaturedpolypeptide,reducedcysteines. Nowdialyzeoutureaandreducingagenttheproteinrefolds.Almost100%activitycomesback. Wegetstheexactproteinback,itrefolded. Principle:Proteinfoldingisaspontaneousevent.Onedoesnotneedanythingelsefortheprotein tofold.Primarystructuremustbeallthatsnecessarytodeterminetertiarystructure. Justusemercaptoethanolandnochaotropeandtheproteinbecomesscrambled.Disulfide bondsarecreatedwheretheyarentsupposed.Yougetinactiveenzyme,becausestructureis distorted.Correctformationofproteinstructureisimportantforitsfunction. Ifyouaddureaandthenmercaptoethanolbacktothedistortedenzyme,itgoesbackinactive shape.Becausenowtheprimarystructurewasallowedtofoldcorrectlyandthedisulfidebonds getbrokenandareallowedtoreforminrightarea. Dynamicstructures Sidechainscanmovearoundonsurface. Wholedomainscanmove. Conformationalflexibilityisknownasbreathing. Moleculardynamicstriestosimulatethisbreathing. Folding Foldingtimeintimeframeseconds. Foldindirectedpathways.Onethatfollowsadecreaseinfreeenergy.Onceasmallpieceof proteinfoldstherestfollowreallyfast.Cooperativeprocess.

Generalsteps: 1)Unfoldedpolypeptidestraightoutofribosome(highentropy) 2)Formationofsecondarystructure(5ms) 3)hydrophobiccollapse,hydrophobicgroupstrytogetawayfromsolvent.Statecalledmolten globalstate 4)Formationofdomains(51000ms) 5)Formationoftertiarystructure(lowentropy).Slowbecausetherearemanydifferenttertiary structureswithsimilarenergiesitcouldbe.Proteintakestimegoingthroughthesetofindthe bestone.Tryingtofindthewaydowntothebottomoffreeenergywell Triestogetlowestenergylevel.Butitcangettrappedinlocalminima.Needsrandomthermal eventtobumpitinrightdirection. Entropyofmoleculegoesdown.Butentropyofsurroundingwatergoesup,becausenowthey canmakemorebondswitheachother. Chaperons Sometimesneedanextracomponent.Typicallywhenyouhaveamisfoldedproteinina confirmationyoucantgetoutof.Exposedhydrophobicgroups.Misfoldedproteinswiththeir hydrophobicgroupssticktogetherinaggregategroups.Theseaggregategroupsareinmany knowndiseases. Eitherdegradethemorallowthemtorefoldwithchaperons. Enzyme:Bindtounfoldedormisfoldedproteinstoproperlyassociatethehydrophobicsegments andthereforepreventitfromformingaggregates. MostchaperonsareATPases Proteindisulfideisomerase Putstogetherdisulfidebridgesinproteinfolding. Correctsincorrectbridges. Oxidizedandthenafterbuildingbridgeisreduced. ATP Highenergyesterbondveryspontaneous.30kJfreeenergy. CleavageATPtoADPorAMPisall30kJ. Givesspontaneitytoreactionsthatmightnotoccurotherwise. GroEL/ESChaperonins Largemoleculecage,14subunitsofGroELunitsand7GroESsubunits(cap). AllGroELcanbindandcleaveATP GroELringcanencloseandfoldproteins(70kDaltons) Cycle

CisringbindstoATP.Thisopensupupcavityandmisfoldedpolypeptidetoenter.Induces bindingofGroEscapontop.Proteincagedin.Shieldsproteinfromcontactwithotherproteins. TheCisringshydrolyzeATPtoADP(10s)andproperlyfoldsit.Secondunfoldedproteincomes infrombottomTransring,including7ATP. Capcomesofftopcorrectlyfoldedproteincomesoutandbottomunfoldedproteincomesupinto cisring. Proteinfoldinggonewrong Alzheimers Plaqueformationsareproteinaggregatesthatareresistanttosolubles.Veryhard togetridof.ProteinscalledAbetaprecursorwhichisafragmentoflargerprotein.Proteincant foldproperly.Plaques(MISSING).Wehavecausation,butnotcorrelation. Prions diseasesareinfectious.(Missing)

Lecture9 Whyuseenzymes? Speedupreactions,ormakereactionshappenthatwouldotherwiseneveroccur. Carryoutmanyofthesereactionsundermildphysiologicalcondition(pHclosetoneutral,low temps,lowpressure,etc.) Mechanismthattightlyregulationofchemicalreactions(ex.hemoglobin) Categorizing Enzymename:[substrate]+ase Specificity Enzymesworkonveryspecificsubstrates. Usehydrophobicinteractions,electrostatics,hydrogenbondstobind. Activesitesiswhereenzymesbindsubstrates.Veryspecificpocket. Verystereospecific.Selectspecificstereoisomer. Additionalfactorsforcatalysis Cofactors BoundMetalions,orcoenzymes Coenzymes Prostheticgroup(alwaysboundstructuretoenzyme),Cosubstrate(bindsand thendissociateswhenreactionisdone. Ethanolreaction EthanolplusNAD+andenzymeADH=>Acetaldehyde+NADH+H+ NAD+actsasprotonshuttle Apoenzyme(inactive)+Cofactor=>Holoenzyme(active)

ActivationEnergy Highfreeenergytolowfreeenergy Transitionstateishigherenergystate,whereitismoreunstable. Thehigherthetransitionstate,ortheenergythemoredifficultitisto overcometransitionstate. Freeenergyofactivation Energyfromreactanttotransitionstate Freeenergyofreaction Energyfromreactanttoproduct Transitionstatehaslowestamountofmoleculesinthereaction. Multistepreactions Highesttransitionstateisratedeterminingstep. Doesntmatterwhattheenergyoftheothertransitionstateis. Catalyse Onlylowerenergyoftransitionstate Actsrateenhancement Acceleratesforwardandreversedreactionbysameamount Notchangingenergyofproductsorsubstrate. Canonlymakespontaneousreactionsgomorequicker,notnonspontaneous. LoweringofactivationenergyistherateenhancementdeltadeltaG Enzymecatalyticmechanism Canusemultiplemechanismsatsametime. Acidcatalysis Stabilizescarbonylgrouptransitionstateviadonating hydrogen Basecatalysis Stabilizeschargebytakinghydrogen EnzymesactiveinverynarrowrangesofpH Covalentcatalysis Usenucleophilestoformcovalentbondsand intermediatespecies. NucleophilesareROH,RSH,RNH3,Imidazolegroup. Themorestabletheintermediatetheharderitisto decomposeitagain.Finetunereactionofthecovalent transitionstatesoitwilldecomposeagain. Metalcations ofenzymes.Usuallytransitionmetalions.Bindtosubstratestoorientthemproperly.2,3+ ionsareverygoodatredoxreactions,byshuttlinghydrogens.Ex.Fe2+,Fe3+,CU2+,Mn2+,Co+.

UsuallynotZinc,becauseitisastructuralmetal. Verygoodatstabilizingorshieldingnegativecharges. ProximityofEnzymes Bringsubstratesclosertoeachother,holdthemintoplaceandforcethemtoreact.Puttingthem inrightorientation.Placereactantsinbestposition. Provideoptimalelectrostaticenvironment. Mostofthecatalyticenhancementoftheratecomesfromholdingandrestrictingconfirmationof substrates.Increaseratetremendouslybyjustputtingtheminrightplace. Mechanisms Enzymesbindmoststronglytotransitionstates,becausebindingsubstrateswouldholditlikeit is.Enzymeisgoingtowanttohavethereactionintransitionstate.Enzymemodelledafter transitionstate.Doesnotbindsubstrate. IthinkEScomplexisEnzymeplussubstrate ESdicescomplexisEnzymeplustransitionstate. Enzymesmaketheactivationenergybelower Inhibitors Moleculesthataretransitionstateanalogs.Targetfordrugdesign,lookforsomethingthatlooks liketransitionstate.

Lecture10Enzymekinetics(Stilldobook) Achemicalreactionhasavelocityandarateconstant A(substrate)>P(product) Rateofreaction:howquicklysubstrategetsconvertedintoproduct.Ordepletionofsubstrate Rate: v:d[P]/dt=d[A]/dt=k[A] RateisdependentonamountofsubstrateatthetimeandhowlikelyitisthatAwillconvertinto P.Thisistherateconstant.Velocitydependentonconcentration. Rateconstant(k) Likelihoodthatreactionwilloccur,orthattwomoleculeswillcollideand react. Ratevelocity(v) rateofappearanceofproductordisappearanceofreactant. Firstorderreaction A>P v=d[A]/dt=k[A]

Velocityproportionalto[A].Unimolecular.

Secondorderreaction 2A>P v=k[A]^2 Pseudofirstorder A+B>P v=k[A][B]

Velocityproportionalto[A]^2.Biomolecular.

Velocityproportionalto[A]ifitisreducedconcentration. Secondorder.However,firstorderinrespecttowhichhas thelowerconcentration.

Rateequation Arateequationdescribestheprogressofareactionasafunctionoftime Rateequationln[A]=ln[A]okt Ao=Initialamount Straightlineforfirstorderreaction InfirstorderhalflifeindependentofconcentrationA0 Secondorderisnonlinear,slope.Rateslowsbecausethereislesschanceofacollision. Rateequation1/[A]=1/[A0]+kt 2A>P1/[A]=1[A]0+kt Halflifedependson[A]o t1/2=1/(k[A]0). A+B>If[B]>>[A]Thereactionisfirstorderwithrespectto[A](pseudofirstorder) Reactionvelocity Whenhaveconcentrationsohighthatratedoesnotincrease,rateisconsideredzerothorder.It isindependentofconcentration.Enzymescanbesubstitutedbymeasuringreactionvelocities. Examplewehaveissucrose+H2Oviafructofuranosidaseglucose+fructose. Zerothorderif[sucrose]>>>[Enzyme] E+S<=>ES>P+E First=isk1,k1isbackwardreaction. First=isk2 IfconcentrationofSislow,k1isratedeterminingstep.IfSishigh,k1iszerothorderandk2is ratelimitingstep. RateofES>P+Eisfirstorderv=k2[ES] RateofE+S<=>ESissecondorderv=k1[E][S]k1[ES]k2[ES] MichaelisMenten MichaelisMentenequationrequirestwoimportantassumptions 1)Thereactionisatequilibrium.Ifk1>>k2thenthereactionwillreachequilibriumquickly.

Ks=k1/k1=E*S/ES 2.Thereactionisatsteadystate S>>ETherateofESproductionisequaltorateofESconsumptiond{ES}/dt=0 Progressofreaction VerydifficulttomeasureEandES Canmeasure[E]total=EandES Rateequation Rateofchemicalreactionusingenzyme (EtotalES)*[S]/ES=((k1)+k2)/k1=Km=MichaelisConstant Itsthemeasureofaffinityofenzymetosubstrate,howeffectiveitbinds. [ES]=([Etotal][S]0/(Km+[S]) Vmax=k2[Etotal] MichaelisMentenEquation v0=Vmax[S]/(Km+[S]) Plot Michaelis(Km)constantissubstrateconcentrationat1/2Vmax Kmissubstrateconcentrationat1/2Vmax,orwhenhalftheenzymaticsitesinvolvedinenzyme process,oraffinityofenzymeforitssubstrate. Kmisuniqueforeachenzymesubstratepair Kcat Doubleenzymedoublerateofreaction.Numberofcatalyticcyclesofenzymesperunittime. kcat=Vmax/Etotal atVmaxkcat=k2 kcat/km=catalyticefficiency40Min when[S]<<Kmsecondorderrateconstant v=(kcat/km)[E][S] kmhigh,affinityforsubstanceverylow kcatverylow,thenenzymeisnotveryefficient. LineweaverBurkplot VerydifficulttogetVmaxcondition.Thatswhydolineweaverburkplot,easiertodeterminevmax.

Noonereallyusesit. Curves IfVmaxislower

Enzymebindingtosubstrate,butnotefficientanymore.Enzymemutant probinvolvedincatalysisnotinbinding. IfKmhigherorlower Enzymeinvolvedinbindingbutnotincatalysis

Lecture11 Vmax Km maximalvelocityofenzymeatsaturatedsubstrateconcentration substrateconcentrationatVmax.LowKmthenaffinityveryhigh,Kmhighthen affinityverylow.Highaffinitythenweneedveryfewmoleculesinreactiontoget halfofenzymeswithsubstrate. Turnoverrate

kcat

Inhibitor Moleculethatinhibitsenzymes. Gooddrug=goodinhibitor Competitiveinhibitors Competeswithsubstrateforsubstratebindingsite.Blockssite,preventsitfrombeingcatalysed. Cantgetridofitbecauseitbindstightlytoit. Resemblesubstratebutarenotreactive. Effectivenessofinhibitorcanteachusaboutsubstraterecognitionsite. Ex.succinatehydroginaseinhibitedbymalonate. Tamiflu(oseltamivir)inhibitorofinfluenzaneuraminidase Ki=verylow. Transitionstateanalogsverygoodinhibitors. Designinibitorthatbindstransitionstate,anditbindstighter.Kiwillbelower. Competitiveinhibitorsreduce[E]availablefor[S].BindstoEandtakesitoutofthereaction.Will affectthekinetics.Competitiveinhibitorisreversible. Ki Presenceofinhibitorwillaffectsteadystatekinetics. Ki=[E][I]/[EI] MichaelisMentenequationforit v0=(Vmax[S])/(alphaKm+[S])

alpha=1+[I]/Ki CompetitiveinhibitorsmakeKmappearverylarge CompetitiveinhibitorsdonotaffectVmax Lineweaverplot 1/Vmaxintersectstayssame.Slopeincreases Uncompetitiveinhibitor Inhibitorbindingtoenzymesubstratecomplex. Uncompetitiveinhibitorsdonotneedtoresemblesubstrate Distortetheactivesitetopreventcatalysis. TakesoutESincomplex,thisitwhatdefinesVmax Ki=[ES][I]/[ESI] MichaelisMenton v0=Vmax[S]/(Km+alpha[S]) MakeKmlooksmaller.PullingESoutofequation,drivingSintothereaction.Lookslike substrateisbindingmuchmoretightly. DecreaseVmax Noncompetitiveinhibitor Inhibitorbindingtobothfreeenzymeandtheenzymesubstratecomplex.Doesntcareinwhat theenzymestateisin. Bindstositeawayfromactivesite. MichaelisMenten v0=(Vmax[S])/(alphaKm+alpha[S]) NoncompetitiveinhibitorsdecreaseVmax DependingonvaluesofKiandKi,Kmseemeitherbiggerorsmaller purecompetitiveinhibitorisunreactivebackwards HIV 1985 1989 1995

FindingofHIVprotease.Responsibleformaturationofcapsidproteins Crystallographystructureofproteasedetermined. RochemakesSaquinavir.Madein6yearssincedetermination.

Lecture12Lysozyme Lysozymescleavesglycosidiclinkagesincellwalls(peptidylglycan) NAG Nacetlyglucosamine(extraacetylgroupat2nd) NAM Nacetlymuramicacid(extraacetylgroupat2nd,andextralactateat3rdcarbon) Lysozymecatalysisrate10^8increase Cleavagesite Substratebindingcleftcanfit6saccharideunits.Cleaveonceeverysixsugars.Recognizes NamNagalterations. PositionsofsugarscalledAF.D(4thsugar)sugarmustadopthalfchairconformationto preventclashes.OnlyDsugarinhalfchairconformation.Higherinenergy.Halfchairmusthelp incatalysis.ProteinblocksC5pointingdowntochairconformation.Residuesinenzymearound theDsugaronlyallowforDsugar. Sidechaininteractions: Manyinteractionswithaminoacidstokeepitinthecorrectconfirmation. Cleavage CleavesC1andO1betweenNag(O1)andNam(C1).NaginchairpositionandNamindistorted position. Enzymearrangedtohavehydrogenbonds. Nonenzymemechanism Inacidiccondition. O1oxygengetsprotonated.Cleavageofacetalbond,oxoniumtransitionstate.Oxoniumionhas resonancestabilizedstate,however,onlywhenitsplanar.Lysozymedonatesprotonand stabilizesoxoniumion. Enzymemechanism LookforaminoacidsnextdoDsugarbondthatwewanttocleave. Glu35surroundedbynonpolarresiduesisprotonated.Needstobedeprotonated.Inacidits deprotonated. Asp52surroundedbypolarresiduesisunprotonated.Protonateit. Botharethetoolstocatalysethereaction. Mechanism 1)Substratebindstoenzyme.BindsNagNamanddistortsNamtohalfchair 2)Becauseacidenvironmentglu35isprotonated.Donatesprotontooxygeninacidcatalysis. Cleavebond.Oxoniumtransitionstateform.Inordertoformitneedstobestabilizedpreferential bindingtotransitionstatecatalysis. 3)Oxoniumresonancestabilized.Asp52helpsstabilize.Electrostaticcatalysis 4)Asp52isanucleophileandattackstomakecovalentintermediate.

5)Watercomesinandrecreatesthesugar.Andaspartategroupleaves. (REDO) Transitionstate UsedfluorineonC2insteadofHandGlninsteadofGlu35toslowdowncovalentintermediate.

Lecture13SerineProtease Proteasescatalyzethecleavageofpeptidebonds. Scissilepeptidebonds SpecificityisintheRn1position Specificitypocketdetermineswhereitsgoingtocleave Reactivesubstrateanalogtolabelsidechainsthatareclosetosubstrateorimportantfor catalysis. Catalytictriad Threeresiduesthatworktogether.Alwaysabsolutelyconservedacross enzymesinspeciesandacrossserineproteins Chymotrypsin Asp102,His57,Ser195.Formhydrogenbondswitheachother.Serine protease Convergentevolutionwithserineproteins Proteasecleavagepoints Trypsin Rn1=positivelychargedresidueRnnotPro Chymotrypsin Rn1=bulkyhydrophobicresidueRnnotPro Elastase Rn1=smallneutralresidueRnnotPro SpecificityisintheRn1position.RnusuallycantbeProbecauseitsbackboneisnot compatible. Specificitycomesabout Specificitypocket,meanstheprimarysequenceimmediatelyaboutscissilebondthatdetermine whichsubstratecangoin Catalytictriads foundbyreactiveanalogs 3reactiveresiduesthatworkinconcept. hardcoreconserved. Chymotrypsin Aspartate,histidine,serine.

Serineproteaseevolution Catalytictriadsdifferentindifferentproteases Differencesdonotcomefromsameancestor

Appearindependentlytocreatesamemechanism Mechanism 1)His57deprotonatesSer195tocreatenucleophile(generalbasecatalysis) 2)Nucleophilicattackcreatesacovalentintermediate(covalentcatalysis) (MECHANISMREDO) bovinetrypsininhibitor.Fitsperfectlyintrypsin.blocksreaction Zymogens Inactiveproteaseprecursors Trypsinogen(inactive),trypsin(active) Canpreventenzymesfrombeingactivetoearly. Cleavageforrapidcascades