大三上课件生化第01章蛋白质2015

StructureandFunctionofproteinsChapter1StructureofhemoglobinFibrousproteinsHistoryofproteinresearchIn1833，amylase(淀粉酶)wasisolatedfrommaltsprout(麦芽)；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(分布广)：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蛋白质的分子组成ProteinsProteinsarenitrogencontainingpolymerscomposedofmanyaminoacidslinkedbyaspecifictypeofcovalentbondspeptidebond.peptidebondElementscompriseofproteinsAllproteinscontainC、H、O、NandSandindividualproteinsmayalsocontainphosphorusormetalelementssuchasiron,zinc,copperoriodine.

组成蛋白质的元素Theaveragenitrogencontentisapproximately16%.

Ifwecandeterminetheamountofnitrogeninonesample,wecankaculateproteincontentofthesamplebasedonthefollowingformular:Theamountofproteinsin100gsample(g%)=Nitrogencontent(g)/sample(g)×6.25×1001/16%Charactersofelementcompositionofproteins各种蛋白质的含氮量很接近，平均为16％。蛋白质元素组成的特点一、Aminoacids

buildingblocksofproteins

Thereareover300kindsofaminoacidinnature,butonly20ofthemcomprisehumanproteinswhicharea-L-aminoacids（exceptglycine）.

Aminoacids-basicstructureH3NCOO-RHCa4. Afewproteinscontainnonstandardaminoacidsthatareformedbypost-translationalmodificationofprolineandlysine.prolineCommonstructureof19AAsa-carbonischiral(asymmetric)exceptinglycine(RisH)2. Aminoacidsaredipolarions(zwitterions[兼性离子])inaqueoussolutionandareamphoteric(兼性的,同时有酸碱性或正负电荷的)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缬氨酸

valineValV

5.96亮氨酸

leucineLeuL

5.98

异亮氨酸

isoleucineIleI

6.02

苯丙氨酸

phenylalaninePheF

5.48脯氨酸

prolineProP

6.30NonpolarandneutralaminoacidsStructureChinesenameEnglishnameOnelettersymbolThreelettersymbol色氨酸

tryptophanTryW

5.89丝氨酸

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

苏氨酸

threonineThrT5.602.Polarandneutralaminoacids天冬氨酸

asparticacidAspD

2.97谷氨酸

glutamicacidGluE

3.22赖氨酸

lysineLysK

9.74精氨酸

arginineArgR

10.76组氨酸

histidineHisH

7.593.Acidicaminoacids4.BasicaminoacidsSpecificaminoacidsProline（亚氨基酸）

Cysteine(半胱氨酸)+CystineDisulferbond-HH胱氨酸（二）Physicalandchemicalpropertiesof20aminoacids1.DipolarionandIsoelectricpoint(两性解离及等电点)Aminoacidsaredipolarions，theextentoftheirionizationisdeterminedbythepHvalueofthesolution.Isoelectricpoint,pIInasolutionwithagivenpHvalue，anaminoacidhasthesametendencyandextentofionizationintocationandanion,andesamphiproticionwhichiselectricallyneutral.ThispHvalueisknownasIsoelectricpoint.pH=pI+OH-pH>pI+H++OH-+H+pH<pIzwitterioncationAnionNonionicandzwitterionicformsofaminoacids.Thenonionicformdoesnotoccurinsignificantamountsinaqueoussolutions.ThezwitterionpredominatesatneutralpH.2.Ultravioletabsorption(紫外吸收性质)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(氨基酸残基).*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

体内存在多种重要的生物活性肽GSHPeroxidaseH2O22GSH2H2OGSSG

GSHReductaseNADPH+H+NADP+(GSH过氧化物酶)(GSH还原酶)

TherearemanyhormonesareoligopeptideorpolypeptideNeuropeptide(神经肽):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

(初原纤维)microfibril

(微管)Themolecularstructureofproteinsinclude:

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

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

Hydrogenbond

多肽链的局部主链构象为蛋白质二级结构Formationofthepeptideplane(肽平面)（一）Peptideunit(肽单元

)6atomsofC

1、C、O、N、H、C

2locatedatthesameplane.Theplaneconstitutedbythe6atomsiscalledpeptideunit.PeptideUnitPeptidebondplane

Maintypesofthe

Secondarystructure

-helix(

-螺旋

)

-pleatedsheet(

-折叠

)

-turn(

-转角

)randomcoil(无规卷曲)

（二）

-Helix（三）

-sheetb-sheet:

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

sectionsruninthesamedirectionAntiparallelbsheet:sectionsrunintheoppositedirection（四）

-turnandrandomcoil

-turnRandomcoil

isusedtoelucidatetheunregularstructuresofthepolypeptide.二个或三个具有二级结构的肽段，在空间上相互接近，形成一个特殊的空间构象，称为模体(motif)

。Twoorthreepeptidefragmentswhichhavesecondarystructuregetclosetoformspecialspatialconformationandiscalledmotif.Motif(orsupersecondarystructure)模体是具有特殊功能的超二级结构α-helix-βturn（orloop）-α-helixmotifβsheet-βturn-βsheetmotifβsheet--α-helix-βsheetmotif

Thecommonformsofmotif

模体常见的形式

Someproteinscontainanorderedorganizationofsecondarystructuresthatformdistinctfunctionalorstructuralmotif.钙结合蛋白中结合钙离子的模体锌指结构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.分子量较大的蛋白质常可折叠成多个结构较为紧密的区域，并各行其功能，称为结构域(domain)。Domainsoffibronectin5Domains分子伴侣(chaperon)通过提供一个保护环境从而加速蛋白质折叠成天然构象或形成四级结构。ChaperonesandproteinfoldingProvideaprotectiveenvironmentforproperfoldingofproteinsHeatshockprotein70(Hsp70):Chaperonin:Hsp60,GrpEL,Hsp10Nucleoplasmin:(核质蛋白)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