人類(lèi)AHI1蛋白SH3結(jié)構(gòu)域的初級(jí)晶體學(xué)研究.pdf

1、中國(guó)科學(xué)技術(shù)大學(xué)碩士學(xué)位論文人類(lèi)AHI1蛋白SH3結(jié)構(gòu)域的初級(jí)晶體學(xué)研究姓名：石珠亮申請(qǐng)學(xué)位級(jí)別：碩士專(zhuān)業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：吳東海20090401ABSTRACTABSTRACTAHI1isacommonhelperprovimsintegrationsiteformurineleukemiasandlymphomasandshowntobecloselylinkedtotheemybproto—oncogeneRecen

2、tlyresearcheshaveshowedthatAHI1callaffecttheproductionofleukemogenandmutationsinAHI1areamaincauseofJoubertSyndrome(JS)RecentlyresearchhasalsoshowedthatAHllbindstightlytohuntingtinassociatedprotein1(HAPl)，whichinvolvedini

3、ntracellulartraffickingandendocytosisofmembranereceptors，andformsastableproteincomplexinthebrainTheinteractionbetweenAHI1andHAP1isimportantfortheearlydevelopmentofthebrainandofferinsightintothepathogenesisofJS，butthedeta

4、ilsofmolecularmechanismareunknownHumanAHllgeneencodesa1，196aminoacidprotein(AHI1orJouberin)，whichiscomprisedofanNterminalcoiledcoileddomain，7WD40repeats，anSH3domain，andseveralpotentialSH3bindingsitesInterestinglytheSH3do

5、mainofAHI1Wasratheruniquesinceithasthekeyconservedresiduesbutshareslittleaminoacidsequenceidentitywiththe“classical”SH3domainsofAbl，F(xiàn)yn，andLck，F(xiàn)urtherinsightcouldbeprovidedifanatomicstructureofAHI1wereavailableAsaninitia

6、lefforttowardsthisgoal，wehavechosentheSH3domain，acommondomainofsignalingmoleculesinvolvedinproteinproteininteractionsasourresearchobjectInthisreport，wehaveclonedandexpressedtherecombinanthumanAhilSH3domainproteinusinganE

7、scherichiacoliexpressionsystemWepurifiedtherecombinantproteintoanapparenthomogenousstateandearlyoutitspreliminarycharacterizationandcrystallizationtrialsWealsoverifiedthepossibilityofSH3domainofAHI1andHAPIinteractionbyGS

8、TpulldownassayRecombinantproteinWaspurifiedinatwo—stepprocedureusingaffinityandsizeexclusionchromatographyandthepuri鑼ofitWasabout95％DLSresultshowtheAHI1SH3domainexistspredominantlyinasingleaggregationstateinsolutionCryst

9、alssuitablefordiffractionexperimentswereobtainedin02MAmmoniumsulfate，O1MSodiumacetatetrihydratepH46，30％喇～Polyethyleneglycolmonomethylether2000byaseating—dropvapordiffusionmethodin20“(2Thecrystaldiffractedto25A。resolution

10、andbelongedtothetetragonalspacegroupP41212，withunitcellparametersa=67377，b=67377，c=98549A，僅咿)，=900，Withonemonomerintheasymmetricunit，theMatthewsvolumewascalculatedtobe194A3Da“1andtheestimatedsolventcontentWas3669％GSTpull