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1、PublishedonWeb12/14/2009MultiparameterScreeningonSlipChipUsedforNanoliterProteinCrystallizationCombiningFreeInterfaceDiffusionandMicrobatchMethodsLiangLi,WenbinDu,*andRustemF.Ismagilov*DepartmentofChemistryandInstituteforBiophysicalDynamics,TheUniVersityofChicago,929East57thStreet,Chica
2、go,Illinois60637ReceivedOctober7,2009;E-mail:feeuoo@uchicago.edu;r-ismagilov@uchicago.eduAbstract:ThispaperdescribestwoSlipChip-basedapproachestoproteincrystallization:aSlipChip-basedfreeinterfacediffusion(FID)methodandaSlipChip-basedcompositemethodthatsimultaneouslyperformsmicrobatchan
3、dFIDcrystallizationmethodsinasingledevice.TheFIDSlipChipwasdesignedtoscreenmultiplereagents,eachatmultiplediffusionequilibrationtimes,andwasvalidatedbyscreeningconditionsforcrystallizationoftwoproteins,enoyl-CoAhydratasefromMycobacteriumtuberculosisanddihydrofolatereductase/thymidylates
4、ynthasefromBabesiabovis,against48differentreagentsat?vedifferentequilibrationtimeseach,consuming12μLofeachproteinforatotalof480experimentsusingthreeSlipChips.ThecompositeSlipChipwasdesignedtoscreenmultiplereagents,eachatmultiplemixingratiosandmultipleequilibrationtimes,andwasvalidatedby
5、screeningconditionsforcrystallizationoftwoproteins,enoyl-CoAhydratasefromMycobacteriumtuberculosisanddihydrofolatereductase/thymidylatesynthasefromBabesiabovis.Topreventcross-contaminationwhilekeepingthesolutionintheneckchannelsforFIDstable,theplatesoftheSlipChipwereetchedwithapatternof
6、nanowells.Thisnanopatternwasusedtoincreasethecontactangleofaqueoussolutionsonthesurfaceofthesilanizedglass.ThecompositeSlipChipincreasedthenumberofsuccessfulcrystallizationconditionsandidenti?edmoreconditionsforcrystallizationthanseparateFIDandmicrobatchscreenings.Crystallizationexperim
7、entswerescaledupinwellplatesusingconditionsidenti?edduringtheSlipChipscreenings,andX-raydiffractiondatawereobtainedtoyieldtheproteinstructureofdihydrofolatereductase/thymidylatesynthaseat1.95?resolution.Thisfree-interfacediffusionapproachprovidesaconvenientandhigh-throughputmet
