人類AHI1蛋白SH3結構域的初級晶體學研究.pdf

1、中國科學技術大學碩士學位論文人類AHI1蛋白SH3結構域的初級晶體學研究姓名：石珠亮申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：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，Fyn，andLck，FurtherinsightcouldbeprovidedifanatomicstructureofAHI1wereavailableAsaninitia

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