老師課件給學(xué)生02 sec bc

SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭SectionB

ProteinStructureB2ProteinstructureB3ProteinAnalysis2014-02-20SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭B2ProteinstructurePrimarystructure(一級(jí)結(jié)構(gòu))Secondarystructure(二級(jí)結(jié)構(gòu))Tertiarystructure(三級(jí)結(jié)構(gòu))Quaternarystructure(四級(jí)結(jié)構(gòu))Domainsandmotifs(結(jié)構(gòu)域與基序)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Primarystructure(一級(jí)結(jié)構(gòu))Definition:

thesequenceofaminoacidsfromtheNtotheCterminusistheprimarystructureofthepolypeptide.Amidebond(酰胺鍵;peptidebond肽鍵)Dipeptide(二肽)Polypeptide(多肽)Sizesforsinglepolypeptidechains:withintherange100-1500aminoacids,thoughlongerandshorteronesexist.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Secondarystructure(二級(jí)結(jié)構(gòu))Definition:Sincetheformationofthehydrogenbonds(C=O???N-H)betweenpeptidebondunitsofthepolypeptidebackbone(多肽鏈骨架),polypeptidesfoldintoseveralregularstructures,include:

-helix(

-螺旋):Thepolypeptidebackboneformsaright-handedhelix(右手螺旋)with3.6aminoacidresiduesperturnsothateachpeptideN-HgroupishydrogenbondedtotheC=Ogroupofthepeptidebondthreeresiduesaway.3.6133.0104.4162.27每圈2.2個(gè)AA每個(gè)氫鍵所封閉的原子數(shù)目SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Secondarystructure(二級(jí)結(jié)構(gòu))

-pleatedsheet(

-折疊片層

-sheet):isformedbyhydrogenbondingofthepeptidebondN－HandC=Ogroupstothecomplementary(互補(bǔ)的)groupsofanothersectionofthepolypeptidechain,includeparallel

-Sheets(正向平行片層)andantiparallel

-Sheets(反向平行片層).SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Tertiarystructure(三級(jí)結(jié)構(gòu))Definition:

Thedifferentsectionsof

-helix,

-sheet,andotherminorsecondarystructuresandconnectingloops(連接環(huán))foldinthreedimensions,itisthetertiarystructureofthepolypeptide.Thebondforces:

Varioustypesofnon-covalentforcesbetweensidechainsholdthetertiarystructuretogether,include:vanderWaalsforces,hydrogenbonds,saltbridges.310每圈3個(gè)AA每個(gè)氫鍵所封閉的原子數(shù)目SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Tertiarystructure(三級(jí)結(jié)構(gòu))Inaddition,covalentdisulfidebonds

(二硫鍵)

canformbetweentwocysteine(半胱)residueswhichmaybefarfromtheprimarystructurebutclosetogetherinthefoldedtertiarystructure.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Quaternarystructure(四級(jí)結(jié)構(gòu))Twoormorepolypeptides膠原質(zhì)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Motifsanddomains(基序與結(jié)構(gòu)域)Motifs(基序:從空間結(jié)構(gòu)上看)Motifsaregroupingsofsecondarystructure.Similarstructuralmotifsarealsofoundinproteinswhichmayhavenosequencesimilarity.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Motifsanddomains(基序與結(jié)構(gòu)域)Domains

(結(jié)構(gòu)域:從功能上看)Domainsformsemi-independent(半自主)structuralandfunctionalunitswithinasinglepolypeptidechain.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭B3ProteinAnalysisProteinpurification(蛋白質(zhì)提純)SizeChargeAffinity(親和力)TwodimensionalelectrophoresisProteinsequencing(蛋白質(zhì)測(cè)序)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Size(大小)Ultra-centrifugation

(超速離心)Itcanbeusedtoseparateproteinsbutisalsoapowerfulanalyticaltoolforstudyingproteinstructure.Gelfiltrationchromatography

(凝膠過濾層析)Thecolumns(柱子)filledwithasuspension(懸浮液)ofbeads(顆粒)containingparticularsizepores－amolecularsieve

(分子篩).Proteinmoleculeslargerthantheporeselute(洗脫)atthebottomrelativelyquickly.Proteinmoleculessmallerthantheporecanenterthebeadsandsohavealargervolumeofbuffer(緩沖液)throughwhichtheycandiffuseandout.Thus,smallerproteinselutefromthecolumnafterlargerones.

SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Charge(電荷)Electrophoresis(電泳):proteinmixtureisappliedtoagelandanelectricfieldacrossthegel.Dependingontheirnetcharge,proteinswilltravelatdifferentratestowardstheanode

(陽極)orcathode

(陰極)andcanberecoveredfromthegelafterseparation.Ion-exchangechromatography(離子交換樹脂):ionsthatareelectro-statically(帶靜電地)boundtotheionexchangerpackedinacolumnarereversibly(可逆地)replacedwithchargedproteinsfromsolution.Then,asaltgradient(鹽濃度梯度)ofincreasingionicstrength(離子強(qiáng)度)ispassedthroughthecolumnandtheboundproteinseluteseparately(分別洗脫).proteinDNA&RNASectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭

Affinity(親和力)Highspecificities(特異性):enzyme-substratereceptor-ligandantibody-antigenForexample:acolumnmadeofasupporttowhichthehormoneinsulin(胰島素)iscovalentlylinkedwillspecificallybindtheinsulinreceptorandnootherprotein.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭FirstdimensionalelectrohporesisSeconddimensionalelectrohporesisSpotspicker

TwodimensionalelectrophoresisSectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Principle-+PI-+WMFirstdimensionalelectrophoresis(IPG)Seconddimensionalelectrophoresis(SDS)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Theexperimentsof2-DEDatabaseSectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Proteinsequencing(蛋白質(zhì)測(cè)序)Proteinsequencing:Applyingaproteinsequencer(蛋白質(zhì)測(cè)序儀):EachpeptideisthensubjectedtosequentialEdmandegradation(連續(xù)Edman降解法)inaproteinsequencer.cDNAsequencing(cDNA測(cè)序)

:mRNAcDNAproteinsequenceThisissimplerandfasterbutitmissespost-translationalmodifications(翻譯后修飾)FrederickSangerreceived佛雷得里克桑革twoNobelprizesforhisworkonproteinsequencingandDNAsequencingSectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭SectionC

PropertiesofNucleicAcidsC1NucleicAcidStructure(核酸的結(jié)構(gòu))C2ChemicalPropertiesofNucleicAcids(核酸的化學(xué)性質(zhì))C3ThermalPropertiesofNucleicAcids(核酸的熱力學(xué)特征)C4DNASupercoiling(DNA超螺旋)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭C1NucleicAcidStructureBases(堿基)Nucleosides(核苷)Nucleotides(核苷酸)Phosphodiester(磷酸二酯鍵)DNAdoublehelix(DNA雙螺旋)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Bases(堿基)Purines

(嘌呤)arebicyclicstructures,include:Adenine(腺嘌呤A)andguanine(鳥嘌呤G)Pyrimidines

(嘧啶)aremonocyclic,include:Cytosine(胞嘧啶),thymine(胸腺嘧啶)anduracil

(尿嘧啶)

TorUThethyminebaseisreplacedbyuracilinRNAThymineis5-methyl-uracilAderine(A)Guanine(G)Thymine(T)NH3OONH3ONH3Uracil(U)Cytosine(C)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Nucleosides(核苷)Nucleosides=Base+Sugar,thebasesarecovalentlyattachedtothe1'-positionofapentosesugar(戊糖).Nucleotides(核苷酸)Nucleotides=Base+Sugar+phosphates(磷酸),inthe5'-positionofsugar,1-3phosphatesmaybeattached.Phosphodiesterbonds(磷酸二酯鍵)Covalentlinkageofaphosphategroupbetweenthe5'-hydroxyl(5-羥基)ofoneriboseandthe3'-hydroxyl(3-羥基)ofthenext.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭DNAdoublehelix(DNA雙螺旋)Discovery:In1953DNAdoublehelixstructurewerededucedbyWatson(34y)andCrick(46y)Structure:TwochainsofDNAareinaright-handeddoublehelix.Thesugar-phosphatebackbonesareontheoutside,andtheplanar(平面的)basesinthecenterofthehelix.Grooves:Betweenthebackbonestrandsrunthemajor(大溝)andminorgrooves(小溝).Basepairs:Thestrandsarejoinedbyhydrogenbondingbetweenthebasesonoppositestrands,toformbasepairs.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭C2ChemicalPropertiesofNucleicAcidsEffectofacid(酸效應(yīng))Effectofalkali(堿效應(yīng))SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Effectofacid(酸效應(yīng))forDNA&RNACompletehydrolysis(完全水解)Instrongacidandathightemperatures,forexampleperchloricacid(HClO4)atmorethan100C,nucleicacidsarehydrolyzedcompletelytotheirconstituents(組分):bases,ribose(ordeoxy-ribose)andphosphate.Partlyhydrolysis(部分水解)Indilute(稀釋的)acid,forexampleatpH3-4,themosteasilyhydrolyzedbondsareselectivelybroken.Apurinichydrolysis:Theglycosylicbonds(糖苷鍵)attachthepurine(嘌呤)basestotheribosebackbone,iftheyarebrokenthenucleicacidesapurinic(脫嘌呤)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭EffectofalkaliforDNA

(堿效應(yīng))DNAdenaturation:Thedouble-strandedstructureoftheDNAbreaksdown;thatistheDNAesdenatured.EffectofalkaliforRNA

(堿效應(yīng))RNAhydrolysis:Inalkali,

thehydrolysisofRNAcomesabout,becauseofthepresenceofthe2‘-OHgroupinRNA,whichisparticipatedinthecleavage(剪切)oftheRNAback-bonebyintra-molecularattack(分子內(nèi)攻擊)onthephosphodiesterbond.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭C3ThermalPropertiesofNucleicAcidsThermaldenaturation(熱變性)Renaturation(復(fù)性)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Thermaldenaturation(熱變性)IncreasedtemperaturecancausethethermaldenaturationofDNAandRNA:RNAdenaturesgradually(逐漸)onheating,butdsDNA‘melts’intosinglestrandsatadefinedtemperature,meltingtemperature(解鏈溫度Tm),Tmisafunction

(函數(shù))ofG+Ccontent

(含量)oftheDNADenaturationmaybedetected

(測(cè)定)bythechangeinA260.

SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Renaturation(復(fù)性)ThethermaldenaturationofDNAmaybereversed(恢復(fù))bycooling(冷卻，酷)thesolution.Thespeedofcoolinghasanimportantinfluenceonthee:FastcoolingallowsonlytoformdsDNAinlocalregions,itisnottheoriginaldsDNAmolecule.Slowcoolingallowsthesamplefullydouble-stranded,withthesameabsorbance(吸光度)astheoriginaldsDNAsample.Therenaturationbetweendifferentnucleicacidstrandsisknownashybridization(雜交).SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭C4DNASupercoilingClosed-circularDNA(閉環(huán)DNA)Supercoiling(超螺旋)Topoisomer(拓?fù)洚悩?gòu)體)Twistandwrithe(纏繞和扭曲)Energyofsupercoiling(超螺旋能)Topoisomerases(拓?fù)洚悩?gòu)酶)SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Closed-circularDNA(閉環(huán)DNA)ManyDNAmoleculesincellsconsistofclosed-circular(閉環(huán))double-strandedmolecules,forexample:bacterialplasmids(質(zhì)粒);bacterialchromosomes(染色體);manyviralDNAmolecules.Thismeansthat:thetwocomplementarysinglestrandsareeachjoinedintocircles,andhasnofreeends;themoleculesaretwisted(纏繞)aroundoneanotherandthetwosinglestrandsarelinked(連接)togetheranumberofturnsinthemolecule.Thisturnnumberisknownasthelinkingnumber

(連接數(shù)，環(huán)繞數(shù)Lk).SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Supercoiling(超螺旋)Supercoiling:ItisthehelixoverthehelixofdsDNA;happensintheclosed-circulardsDNA.Supercoilingdirection:Positive:thetwistisinsamedirectionasthedoublehelix(加緊);Negative:thetwistisinopposite(相反)directionasthehelix(放松).Lk

andLkLk

:thevalueforarelaxed(松弛)closedcircle;Lk:Lk=Lk-Lk

,definedasthenumberof360

twists(纏繞)introducedbeforeringclosure(關(guān)閉).Itquantifies(計(jì)量)the

levelofsupercoiling.Example:MostnaturalDNAisnegativelysupercoiledDNAwhenisolatedfromcellsiscommonlynegatively(-)supercoiledbyaround6turnsper100turns,thatis

Lk/Lk

=-6/100=-0.06.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭PositiveNegativeSectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Topoisomer(拓?fù)洚悩?gòu)體)TheLkisatopological(拓?fù)涞?propertyofaclosed-circularDNA;ThelinkingnumbercannotbechangedwithoutbreakingoneorbothoftheDNAbackbones.Amoleculeofagivenlinkingnumberisknownasatopoisomer(拓?fù)洚悩?gòu)體).Topoisomersdifferfromeachotheronlyintheirlinkingnumber.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Twistandwrithe(纏繞和扭曲)Topologicalchanges(拓?fù)鋵W(xué)改變)Theconformation(構(gòu)象)oftheDNAcanbealtered(改變)whiletheLkremainsconstant,correspondingtothetypesofthesupercoiling(

Lk),theDNAmaybe:Completelyintotwist(纏繞);Completelyintowrithe(扭曲);Commonsituationisbetweenthetwoextremes(極端).

Twand

Wr:

Tw:Twist(纏繞)linkingnumber

Wr:Writhe(扭曲)linkingnumber

Lk=

Tw+

Wr:

Lkmustbeaninteger(整數(shù)),but

Twand

Wrneednot.SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Twistandwrithe(纏繞和扭曲)Twist纏繞：描述兩條單鏈之間的互相作用狀態(tài)；Writhe扭曲：描述兩條雙鏈（四條單鏈）之間的互相作用狀態(tài)。SectionB:ProteinStructureSectionC:PropertiesofNucleicAcids華中師范大學(xué)生科院楊旭Twistandwrithe(纏繞和扭曲)Twist纏繞：描述兩條單鏈之間的互相作用狀態(tài)；Writhe扭曲：描述兩條雙鏈（四條單鏈）之間的互相作用狀態(tài)。SectionB:ProteinStructureSecti