Selectorisarapid,highthroughputassaythatexploresdetergentspacetoidentifythosebestsuitedtostABIlizethemembraneproteinofinterestinadvanceofdownstreamproteincrystallizationtrials.Theassayusesdifferentialfiltrationin94differentdetergentstogenerateinformationregardingboththestabilityandthesizeoftheprotein-detergentcomplex.Thestepsofthisassayaresimple:First,theproteinisboundtoanaffinityresin,washedandelutedinanewdetergent.Theeluateisthenpassedthroughtwofilterplateswithdifferingmolecularweightcut-offs(MWCO).Thefiltrationprofilesprovidestabilityandrelativesizinginformationthatallowstheselectionofdetergentsmostlikelytostabilizeaproteininamannerthatwouldbecommensuratewithcrystallizationandstructuredetermination.

WhyshouldIexploresuchavarietyofdetergents?IntegralmembraneproteinspresentauniquechallengetoproteinchemistsandstructuralBIOLOGists.Intheirnaturalenvironment,theseproteinsexistwithinamosaiclipidbilayerthatisadynamicandcomplexenvironment.Priortostructuralstudy,however,thesemustbeextractedfromthecellmembraneandsolubilizedinsuchawayastosatisfythehydrophobicnatureofthetransmembraneregionswhileensuringthathydrophilicdomainsareincontactwithanaqueousphase.Whileasignificantnumberofdetergentsmaybecapableofkeepingtheproteininasolubleform,thestability,homogeneityandactivityofanygivenmembraneproteincanbetremendouslyaffectedbythepropertiesofthesolubilizingcomponent.

DepositstotheProteinDataBankformembraneproteinscomprisebroadrangeofdetergentanddetergent-lipidcomplexesaspartoftheproteinpreparationsleADIngthediffraction-qualitycrystals.Thus,whilecertaindetergents(DM,DDMorOG,forexample)areknownfortheirsuccessinstabilizingintegralmembraneproteinsforstructuralstudiesandarereportedathighfrequency,significanteffortwouldberequiredtoexploredetergentspace,eventocoverdetergentspreviouslysuccessfulforproteincrystallization.

WHATDOESTHISKITCONTAIN?

1xdetergentscreeningplatecontaining150µlof94detergentsat2xworkingconcentration,oneblankandonepositionforthedetergentcurrentlystabilizingtheproteinsampletobescreened

1x0.22µmfilterplatewithreceptacleplatefordetergentexchange

1xreceptacleplateintowhichtheproteinsandnewlyexchangeddetergentswillbeeluted

1x300MWCOfilterplatewithmatchedreceptacleplate