大三上課件生化第01章蛋白質(zhì)2015

StructureandFunctionofproteinsChapter1StructureofhemoglobinFibrousproteinsHistoryofproteinresearchIn1833，amylase(淀粉酶)wasisolatedfrommaltsprout(麥芽)；andthenpepsin(胃蛋白酶)wasisolatedfromgastricfluid(胃液).In1864，hemoglobinwasisolatedandcrystallized.Attheendof19thcentury，aminoacidscomprisingproteinwaselucidatedandmanytypesofshortpeptidearesynthesized.FrederickSangerDeterminationofthestructureofproteins.Thesestudiesresultedinthedeterminationofthestructureofinsulin.HewontwiceNobelprizein1958and1980.Hedevelopedfurthermethodsforstudyingproteinsandparticularlytheactivecentersofsomeenzymes.HedevelopedmethodsfordeterminingsmallsequencesinRNA.Theworkculminatedinthedevelopmentofthe"dideoxy"techniqueforDNAsequencingaround1975.

Themethodhasbeenimprovedandautomatedinotherhumangenome(3billionnucleotides).Inthemiddleof20thcentury，differentkindsofproteinanalysistechnologyhavebeensetupwhichpromotedtherapiddevelopmentofproteinresearch.In1962，determinedthequaternary

structureofhemoglobin.In1990s，functionalgenomicstudyandproteomicresearchhavebeendeveloped.Atthebeginingof20thcentury，thesecondarystructureofproteinswasdiscovered.Theprimarystructureofproteinswasdetermined.一、BiologicalsignificanceofproteinsProteinsareimportantcomponentsoforganismBroaddistribution(分布廣)：Allorgansandtissuescontainproteins；Theydistributeinallpartsoflivingcells.Highcontent(含量高)：Proteinsaremostabundantorganicmoleculesinlivingcells.Theycomprise45%dryweightofahumanbodyandevenreach80%insometissuessuchaslungandspleen.1）Serveasbiologicalcatalysts（enzymes）2）Regulationofmetabolism(hormones)3）Immunizingprotection(antibodies)4）Transportationandstorageofsomecompounds(hemoglobin)5）Movementandsupport(microfibril)6）Takepartinsignaltransductionpathway(receptor,kinase)2.Proteinsperformimportantbiologicalfunctions3.ProteinscanalsoprovideenergybyoxidationTheMolecularComponentsofProteinSection1蛋白質(zhì)的分子組成ProteinsProteinsarenitrogencontainingpolymerscomposedofmanyaminoacidslinkedbyaspecifictypeofcovalentbondspeptidebond.peptidebondElementscompriseofproteinsAllproteinscontainC、H、O、NandSandindividualproteinsmayalsocontainphosphorusormetalelementssuchasiron,zinc,copperoriodine.

組成蛋白質(zhì)的元素Theaveragenitrogencontentisapproximately16%.

Ifwecandeterminetheamountofnitrogeninonesample,wecankaculateproteincontentofthesamplebasedonthefollowingformular:Theamountofproteinsin100gsample(g%)=Nitrogencontent(g)/sample(g)×6.25×1001/16%Charactersofelementcompositionofproteins各種蛋白質(zhì)的含氮量很接近，平均為16％。蛋白質(zhì)元素組成的特點(diǎn)一、Aminoacids

buildingblocksofproteins

Thereareover300kindsofaminoacidinnature,butonly20ofthemcomprisehumanproteinswhicharea-L-aminoacids（exceptglycine）.

Aminoacids-basicstructureH3NCOO-RHCa4. Afewproteinscontainnonstandardaminoacidsthatareformedbypost-translationalmodificationofprolineandlysine.prolineCommonstructureof19AAsa-carbonischiral(asymmetric)exceptinglycine(RisH)2. Aminoacidsaredipolarions(zwitterions[兼性離子])inaqueoussolutionandareamphoteric(兼性的,同時(shí)有酸堿性或正負(fù)電荷的)3. Thesidechains

(R)differinsize,shape,chargeandchemicalreactivityHglucineCH3analineL-generalformularofaminoacidsRC+NH3COO-HGeneralformularofaminoacids（一）ClassificationofaminoacidsAminoacidscontainingaliphatichydrocarbon.Aminoacidscontaininghydroxylgroup.Aminoacidscontainingacidicgrouporderivativesofaminogroup.Aminoacidscontainingbasicgroup.Aminoacidscontainingaromaticgroup.Non-polaraminoacids:Aliphatic

aminoacids;

Aromaticaminoacids;Iminoaminoacids.2.Polaraminoacids:(1)Neutralaminoacids(2)Acidicaminoacids(3)Basicaminoacids*TheEnglishname,abbreviationletterandclassificationof20kindsofaminoacidsareasfollowing;甘氨酸

glycine

Gly

G

5.97丙氨酸

alanineAlaA

6.00纈氨酸

valineValV

5.96亮氨酸

leucineLeuL

5.98

異亮氨酸

isoleucineIleI

6.02

苯丙氨酸

phenylalaninePheF

5.48脯氨酸

prolineProP

6.30NonpolarandneutralaminoacidsStructureChinesenameEnglishnameOnelettersymbolThreelettersymbol色氨酸

tryptophanTryW

5.89絲氨酸

serineSerS

5.68酪氨酸

tyrosineTryY

5.66

半胱氨酸

cysteineCysC

5.07

蛋氨酸

methionine

MetM

5.74天冬酰胺

asparagineAsnN

5.41

谷氨酰胺

glutamineGlnQ

5.65

蘇氨酸

threonineThrT5.602.Polarandneutralaminoacids天冬氨酸

asparticacidAspD

2.97谷氨酸

glutamicacidGluE

3.22賴氨酸

lysineLysK

9.74精氨酸

arginineArgR

10.76組氨酸

histidineHisH

7.593.Acidicaminoacids4.BasicaminoacidsSpecificaminoacidsProline（亞氨基酸）

Cysteine(半胱氨酸)+CystineDisulferbond-HH胱氨酸（二）Physicalandchemicalpropertiesof20aminoacids1.DipolarionandIsoelectricpoint(兩性解離及等電點(diǎn))Aminoacidsaredipolarions，theextentoftheirionizationisdeterminedbythepHvalueofthesolution.Isoelectricpoint,pIInasolutionwithagivenpHvalue，anaminoacidhasthesametendencyandextentofionizationintocationandanion,andesamphiproticionwhichiselectricallyneutral.ThispHvalueisknownasIsoelectricpoint.pH=pI+OH-pH>pI+H++OH-+H+pH<pIzwitterioncationAnionNonionicandzwitterionicformsofaminoacids.Thenonionicformdoesnotoccurinsignificantamountsinaqueoussolutions.ThezwitterionpredominatesatneutralpH.2.Ultravioletabsorption(紫外吸收性質(zhì))Mostproteinscontaintheabovetwotypesofaminoacids,sothepropertycanbeusedforthequantificationofproteinsbymeasuringtheabsorbanceatthewavelengthof

280nm

.Absorptionofultravioletlightbyaromaticaminoacids.Tryptophan(色氨酸)andtyrosine(酪氨酸),absorbultravioletlight.Thepeakofabsorbanceofthemisaroundthewavelengthof

280nm

.3.Reactionwithninhydrin(茚三酮)Aminoacidsreactwithninhydrintoformapurplecompoundwithpeakabsorbanceoflightatthewavelengthof570nm.Itisusedtoquatitativeanalysisofaminoacidsbymeasuringtheamountoflightabsorbedbytheninhydrinderivative..二、Peptide*Peptidebondisanamidelinkageformedbydehydrationandcondensationofthe

-carboxylgroupofoneaminoacidandthe

-aminogroupofanotheraminoacid.（一）Peptide

Peptideisacompoundcomposedofaminoacidslinkedbypeptidebonds.+-HOHGlycylglycinePeptidebobdGlycineGlycine甘氨酰甘氨酸Differenttypesofpeptide*Twoaminoacidscanbecovalentlyjoinedthroughapeptidebondtoform

adipeptide(二肽)andthreeaminoacidsformatripeptides(三肽)andsoon.*

Anaminoacidunitinapeptideisoftencalledaresidue(氨基酸殘基).*Whenafewaminoacidsarejoinedinthisfashion,thestructureiscalledanoligopeptide(寡肽).

Whenmanyaminoacidsarelinkedtogether,theproductiscalledapolypeptide(多肽).Nterminal(N末端)：Inapeptide,theaminoacidresidueattheendwithafreeα-aminogroupistheamino-terminal(orN-terminal)residue;Cterminal

(C末端)：

Inapeptide,theaminoacidresidueattheotherend,whichhasafreecarboxylgroup,isthecarboxyl-terminal(C-terminal)residue.Polypeptidechainhastwoterminals*Polypeptidechainisamolecularstructurethatisformedbymanyaminoacidslinkedwithpeptidebond.N-terminalC-terminalBovineribonuclease牛核糖核酸酶1.Glutathione,GSH(谷胱甘肽)GlutamineGlycineCysteineGlutamyl-cysteinyl-glycine（二）Afewkindsof

biologicallyimportantActivePeptides

體內(nèi)存在多種重要的生物活性肽GSHPeroxidaseH2O22GSH2H2OGSSG

GSHReductaseNADPH+H+NADP+(GSH過氧化物酶)(GSH還原酶)

TherearemanyhormonesareoligopeptideorpolypeptideNeuropeptide(神經(jīng)肽):endorphins(31aminoacids)2.Polypeptidehormonesandneuropeptide

TheMolecularStructureofProteins

Section2Humansmakeabout100,000differentproteinsfrom25,000genes?

FredSanger-1958Nobelprizefordeterminingaasequence

ofINSULIN

Averageproteincontains300to400aa's

andhasaMWof30,000to45,000d

Proteinsmostoftenhasaglobular(spherical)3-Dshape

and

is

negativelychargedGeneralknowledgeofproteins一、Classificationofproteins*Accordingtothecomponents:

SimpleproteinConjugatedprotein=Protein+Non-proteingroup*Accordingtotheshapeofprotein:FibrousproteinGlobularproteinGlobularproteins:enzymesComplementaryfitofasubstratemoleculetothecatalyticsiteonanenzymemolecule.

Proteinstructure－ShapesFibrousproteins:importantstructuralproteins(silkfibroin,keratininhairandwools)keratininhairKeratin

(角蛋白)Protofibril

(初原纖維)microfibril

(微管)Themolecularstructureofproteinsinclude:

PrimarystructureSecondarystructureTertiarystructureQuaternarystructureSpatialstructureStructureofhemoglobinTheprimarystructureofproteinsreferstonumberandorderofthelineraminoacidresiduesincludingthelocationofanydisulfidebones(二硫鍵).Itistheaminoacidsequenceofthepolypeptide.一、PrimarystructureofproteinsChemicalbonds:

Peptidebond，disulfidebondsforsomeproteinsPrimarystructure:StructureofthepentapeptideSer-Gly-Tyr-Ala-Leu.NterminusCterminusPrimarystructure:Primarystructuredeterminesthespatialstructureofproteinandaffectproteinfunction.Primarystructureofbovineinsulin二、Secondarystructureofproteinsistheregularfoldingofthepolypeptide“backbone”,withoutconsideringthesidechain.Secondarystructure:Mainchemicalbond：

Hydrogenbond

多肽鏈的局部主鏈構(gòu)象為蛋白質(zhì)二級結(jié)構(gòu)Formationofthepeptideplane(肽平面)（一）Peptideunit(肽單元

)6atomsofC

1、C、O、N、H、C

2locatedatthesameplane.Theplaneconstitutedbythe6atomsiscalledpeptideunit.PeptideUnitPeptidebondplane

Maintypesofthe

Secondarystructure

-helix(

-螺旋

)

-pleatedsheet(

-折疊

)

-turn(

-轉(zhuǎn)角

)randomcoil(無規(guī)卷曲)

（二）

-Helix（三）

-sheetb-sheet:

hydrogenbondingofthepeptidebondN-HandC=OgroupstothecomplementarygroupsofanothersectionofthepolypeptidechainAstereo,space-fillingrepresentationofthesix-strandedantiparallelbsheet.Parallelbsheet:

sectionsruninthesamedirectionAntiparallelbsheet:sectionsrunintheoppositedirection（四）

-turnandrandomcoil

-turnRandomcoil

isusedtoelucidatetheunregularstructuresofthepolypeptide.二個(gè)或三個(gè)具有二級結(jié)構(gòu)的肽段，在空間上相互接近，形成一個(gè)特殊的空間構(gòu)象，稱為模體(motif)

。Twoorthreepeptidefragmentswhichhavesecondarystructuregetclosetoformspecialspatialconformationandiscalledmotif.Motif(orsupersecondarystructure)模體是具有特殊功能的超二級結(jié)構(gòu)α-helix-βturn（orloop）-α-helixmotifβsheet-βturn-βsheetmotifβsheet--α-helix-βsheetmotif

Thecommonformsofmotif

模體常見的形式

Someproteinscontainanorderedorganizationofsecondarystructuresthatformdistinctfunctionalorstructuralmotif.鈣結(jié)合蛋白中結(jié)合鈣離子的模體鋅指結(jié)構(gòu)Motif三、TertiarystructureofproteinsHydrophobicinteraction、saltbond、hydrogenbondandVanderWaalsforces.MainchemicalbondsThedifferentsectionsof

-helix,

-sheet,andotherminorsecondarystructureofapolypeptidefoldinthreedimensionsandformTertiarystructure.Itindicatesthearrangementofallatomsofapolypeptideinspatialstructure.（一）Tertiarystructure

α-helixβ-sheetβ-turnRandomcoilMyoglobin(Mb)N-terminalC-terminalHydrogenbond-Hydrophobicinteraction-linkinteriorresiduesSaltbond-linksurfaceresiduesDisulfidebondVanderwaalsforcesVariousforcesstabilizeproteinstructureDomainoffebrinectin（二）LargepeptideshavedistinctdomainsDomainsarethefundamentalfunctionalandthreedimensionalstructuralunitsofthepolypeptide.分子量較大的蛋白質(zhì)常可折疊成多個(gè)結(jié)構(gòu)較為緊密的區(qū)域，并各行其功能，稱為結(jié)構(gòu)域(domain)。Domainsoffibronectin5Domains分子伴侶(chaperon)通過提供一個(gè)保護(hù)環(huán)境從而加速蛋白質(zhì)折疊成天然構(gòu)象或形成四級結(jié)構(gòu)。ChaperonesandproteinfoldingProvideaprotectiveenvironmentforproperfoldingofproteinsHeatshockprotein70(Hsp70):Chaperonin:Hsp60,GrpEL,Hsp10Nucleoplasmin:(核質(zhì)蛋白)ClassificationRoleofchaperonesinproteinfoldingProvideaprotectedenvironmentforproperfoldingofproteinsCorrectthemisfoldingofproteinsThemainforceofcombinationamongdifferentsubunitsishydrogenbondandionbond.四、Quaternarystructure

Thesesubunitsmaybeidenticalordifferent.Thesameforceswhichstabilizetertiarystructureholdthesesubunitstogether.Thisleveloforganizationcalledquaternarystructure.Manyproteinsarecomposedoftwoormorepolypeptidechains(subunits).

Eachofthem

hastertiarystructureandiscalledsubunit.QuaternaryQuaternarystructureofhemoglobinAdvantagesofthequaternarystructure:Itallowsverylargeproteinmoleculestobemade,suchastubulin.Itcanprovidegreaterfunctionalitytoaproteinbycombiningdifferentactivitiesintoasingleentity.TheinteractionsbetweenthesubunitscanoftenbemodifiedbybindingofsmallmoleculesandleadtotheallostericeffectsseeninenzymeregulationTheRelationshipBetweenStructureandFunctionofProteinsSectionthree（一）Primarystructureisthefoundationofthespatialstructure一、TheRelationshipBetweenStructureandFunctionofproteinsPrimarystructureofbovineDisulfidebondNativestate，catalyticactivityUrea、β-mercaptoethanolRemoveurea、β-mercaptoethanolUnfoldingstate，inactive（二）Correlationbetweenprimarystructureandproteinfunctione.g：sicklecellanemiaN-val·his·leu·thr·pro·glu·glu·····C(146)HbSβpeptidechainHbAβpeptidechainN-val·his·leu·thr·pro·val

·glu·····C(146)二、TheRelationshipBetweenSpatialStructureandFunctionofproteins（一）Structureofmyoglobinandhemoglobin（三）TheconformationalchangeofproteinsanddiseaseEventhereisnochangeofprimarystructureofaprotein,theimproperfoldingorconformationalchangeoftheproteinalsocanleadtodisease.Huntintondisease,AD,madcowsyndrome.TheconformationalchangeofproteinsinmadcowdiseaseMadcowdiseaseisakindofneurodegenerativediseaseofhumanandanimalscausedbyprionprotein,(PrP).NormalPrPisaboundinα-helix，isknownasPrPc.PrPccanbeconvertedintoproteinPrPsccontainingmoreβ-sheetstructureaffectedbysomeunidentifiedproteins，andresultsinsufferingdisease.PrPcα-HelixPrPscβ-sheetNormalMadcowdiseaseSection4ThePhysicalandChemicalCharacters,andSeparationandPurificationofProteins（一）Proteinsarezwitterions

一、Physicochemicalpropertiesofprotein

Mostproteinshavelargenumberofionizablegroupssuchassidechainofaminoacids,theN-andC-terminus.AtagivenpH,aproteincanbepositively-chargedornegtively-charged.Isoelectricpoint,(pI)pHwherethereisnonetchargeinmolecule

AproteinalsohasitspI.AtitspI,aproteinbearsnonetcharge.CanyouguessthepIvalueofaacidicprotein?Acidicorbasic?（二）Proteins

are

ColloidmoleculesProteinsaremacromolecules.Themolecularweightofproteinisbetween104and106kD，andthediametercanreachcollidrange，1～100nm.*Chargesonthesurfaceofproteinsandwaterlayerarethetwostabilityfactorstopreventproteinaggregationinsolution.+++++++Postively-charged－－－－－－－－NegativelychargedProteinatpIWaterlayer++++++++Postively-Chargedprotein－－－－－－－－Negatively-ChargedproteinUnstableproteinparticlesacidbaseacidbaseacidbasedehydationdehydationdehydationCongregationofproteininsolution（三）Denaturation,precipitationand

coagulation*Denaturationofproteins

istheprocessinwhichaproteinorpolypeptideistransformedfromanorderedtoadisorderedstatewithoutruptureofcovalentbonds

Anyproteincanbedenaturedundersomeconditions.Somedenaturantscandisruptthesecondary,tertiaryandquaternarystructureofproteins.Thisleadstothechangeofphysicochemicalcharactersoftheproteinandlossofbiologicalfunctionoftheprotein.

Denaturingagentsinclude:

heat、organicsolvents(ethanol)、strongacidorbase、heavymetalion(lead,mercury),detergentandalkaloidreagent.

Essenceofdenaturation——

destroynoncovalentbondand

disulfidebond，withoutchangingprimarystructureofproteins.

Application:KillbacteriaEffectivestorageofproteinManydenaturedproteinsrefoldsspontaneouslyinvitrowithpanyingrestorationofbiologicalactivity.Itisknownasrenaturation.DenaturatedproteinscanberenaturatedNativestate，catalyticactivityUrea、β-mercaptoethanolRemoveurea、β-mercaptoethanolUnfoldingstate，inactiveProteinmoleculesaggregatetogetherandsettleoutofthesolution.Precipitationisusuallyinducedbyadditionofasaltoranorganicsolvent,orbychangingthepHtoalterthenatureofthesolution.Theyarenotalwaysdenaturatedwhentheyprecipitatedfromthesolution.What’sprecipitationofprotein?What’scoagulationofprotein?Coagulation

istherandomaggregationofalreadydenaturedproteinmolecules.

Thecoagulumisoftenturbid,andtheformationofthecoagulumisusuallythermallyirreversible.Acoagulummaysettleoutofsolution.

（四）UltravioletabsorptionBecauseproteinscontainaromaticacidssuchastryptophan,tyrosine,phenylalanine,thealsohavetheabilitytoabsorbultravioletlightatthewavelengthof280nm.Thispropertyofproteinscanbeusedforquantitativeanalysisofproteins.（五）Colorreactionofproteins⒈NinhydrinreactionProteinscanbehydrolyzedbyacidorbase,orproteolyticenzymestoproduceaminoacids.⒉BiuretreactionIfproteinsandpolypeptidesareheatedtogetherwithbluestone(coppersulfate)indilutealkalinesolution，theywillformapurpleorredcompound.Thisiscalledbiuretreaction.Itcanbeusedtodetecttheextentofthehydrolysis.MyoglobinandHemoglobinTheyhaveabilitytobindmolecularoxygen(carryoxygen)MyoglobinismonomericandhemoglobinistetramericMyoglobinstoresoxygenandhemoglobintransportsoxygenSection5StructureofhemoglobinO2（一）Oxygen-bindingcurvesofhemoglobinandmyoglobin（二）OxygenationofhemoglobininvolvesconformationalchangesThequaternarystructureofhemoglobinleadstophysiologicallyimportantallostericinteractionsbetweenthesubunits.Conformationalchangeatonesubnuitsleadtoanewsetofbindinginteractionsbetweenadjacentsubunits.Sobindingofthefirstoxygenfacilitatethebindingoftheremainingthreeoxygenmolecules.pI,primarystructure,chemicalbonds,domain,peptideunitImportantconcepts:Keypoints:Propertiesofaminoacidsandproteins.Structuresofproteinsandchemicalbondsforstabilizationofstructuresofproteins

Relationshipbetweenstructureandfunction.B3 Proteinanalysis1.

Purification:toobtainenoughpuresampleforstudy2.Sequencing:determinetheprimarystructureofapureproteinsample

3.Massdetermination:determinethemolecularweight(MW)ofan interestedprotein.

4.X-raycrystallographyandNMR:determinethetertiarystructureofagivensample.ProteinpurificationTopurifytheinterestedproteinfromotherproteinsandnon-proteinmoleculesexistinginthecellsAnessentialexperimentalsteppriortostudyofanyindividualproteinTheprincipalpropertiesofproteinsusedforpurificationSize:gelfiltrationchromatography2.Charge:ion-exchangechromatography,isoelectricfocusing,electrophoresis3.

Hydrophobicity:hydrophobicinteractionchromatography4.Affinity:affinitychromatography5.binanttechniques:involvingDNAmanipulationandmakingproteinpurificationsoeasyGelfiltrationchromatographyProteinstructureBead:

thematrixdeterminethesizeofthepore

ProteinstructureUltracentrifugation:

canbeusedtoseparateproteinsaccordingtotheirsizeandshapethatdeterminetheirsedimentationrate.Verylargeproteincomplex.Proteinstructure2.Charge:

ion-exchangechromatography,isoelectricfocusing,electrophoresisIsoelectricpoint(pI):thepHatwhichthenetsurfacechargeofaproteiniszero.--------++++++++pH=pIpH<pIpH>pIProteinstructureCharge1:Ion-exchangechromatographyColumn+anions+++SamplemixtureProteinbindingColumn+proteinsColumn+anionsIondisplacingPurifiedproteinProteinstructure+Charge2:ElectrophoresisProteinmigrateatdifferentpositiondependingontheirnetchargeProteinstructureCharge3:IsoelectricfocusingAproteinwillstopmovingatpositioncorrespondingtoitsisoelectricpoint(pI)inapHgradientgel.Proteinstructure3.Hydrophobicity:hydrophobicinteractionchromatographySimilartoion-exchangechromatographyexceptthatcolumnmaterialcontainsaromaticoraliphaticalkylgroupsProteinstructure4.AffinitychromatographyEnzyme-substratebindingReceptor-ligandbindingAntibody-antigenbindingProteinstructure5.binanttechniques:Clonetheinterestedproteinencoding

geneinanexpressionvectorwithapurificationta