Protein Crystallization相关交流-蚂蚁淘在线

Background:

Proteins,likemanymolecules,canbepromptedtoformcrystalswhenplacedintheappropriateconditions.Inordertocrystallizeaprotein,thepurifiedproteinundergoesslowprecipitationfromanaqueoussolution.Asaresult,individualproteinmoleculesalignthemselvesinarepeatingseriesof"unitcells"byadoptingaconsistentorientation.Thecrystalline“lattice”(Fig.1)thatformsisheldtogetherbynoncovalentinteractions(Rhodes,1993).TheimportanceofproteincrystallizationisthatitservesasthebasisforX-raycrystallography,whereinacrystallizedproteinisusedtodeterminetheprotein’sthree-dimensionalstructureviaX-raydiffraction.In1934,BernalandHodgkindiscoveredthatproteincrystalssurroundedbytheirmotherliquorgavebetterdiffractionpatternsthandriedcrystals.Usingpepsin(Fig.2),theywerethefirsttodiscernthediffractionpatternofawet,globularprotein.PriortoBernalandHodgkin,proteincrystallographyhadonlybeenperformedindryconditionswithinconsistentandunreliableresults(Tulinsky,1999).

Figure1.Cartoonofahypotheticalcrystallinelattice.Colorshavebeenaddedmerelyasreferencepoints.

Initialprogresswasslowindiscerningproteinshapes,duetothedifficultyinherentincrystallizingproteins.WhentheProteinDataBank(http://www.pdb.org)wasfoundedin1971,itcontainedonlysevenstructures(Berman,etal.,2000).Sincethen,thepaceatwhichproteinstructuresarebeingdiscoveredhasgrownexponentially,withthePDBcurrentlycontainingover20,000structures(PDB,2003).OurABIlitytocrystallizeproteinshasobviouslyimproved,andthiswebpagehighlightsafewofthemethodscurrentlyavailable.

Figure2.Three-dimensionalstructureofporcinepepsin.Pepsinwasthefirstglobularproteincrystalusedsuccessfullyinx-raydiffraction.ImagecourtesyofProteinDatabank(ID:3PSG;PDB,2003).

Overview:

Thegoalofcrystallizationisusuallytoproduceawell-orderedcrystalthatislackingincontaminantsandlargeenoughtoprovideadiffractionpatternwhenhitwithx-ray.Thisdiffractionpatterncanthenbeanalyzedtodiscerntheprotein’sthree-dimensionalstructure.Proteincrystallizationisinherentlydifficultbecauseofthefragilenatureofproteincrystals.Proteinshaveirregularlyshapedsurfaces,whichresultsintheformationoflargechannelswithinanyproteincrystal.Therefore,thenoncovalentbondsthatholdtogetherthelatticemustoftenbeformedthroughseverallayersofsolventmolecules(Rhodes,1993).Inadditiontoovercomingtheinherentfragilityofproteincrystals,thesuccessfulproductionofx-rayworthycrystalsisdependentuponanumberofenvironmentalfactorsbecausesomuchvariationexistsamongproteins,witheachindividualrequiringuniqueconditionsforsuccessfulcrystallization.Therefore,attemptingtocrystallizeaproteinwithoutaprovenprotocolcanbeverytedious.Somefactorsthatrequireconsiderationareproteinpurity,pH,concentrationofprotein,temperature,andprecipitants.Inorderforsufficienthomogeneity,theproteinshouldusuallybeatleast97%pure.pHconditionsarealsoveryimportant,asdifferentpH’scanresultindifferentpackingorientations.Buffers,suchasTris-HCl,areoftennecessaryforthemaintenanceofaparticularpH(BrandenandTooze,1999).Precipitants,suchasammoniumsulfateorpolyethyleneglycol,arecompoundsthatcausetheproteintoprecipitateoutofsolution(Rhodes,1993).

VaporDiffusion:

Twoofthemostcommonlyusedmethodsforproteincrystallizationfallunderthecategoryofvapordiffusion.Theseareknownasthehangingdropandsittingdropmethods.Bothentailadropletcontainingpurifiedprotein,buffer,andprecipitantbeingallowedtoequilibratewithalargerreservoircontainingsimilarbuffersandprecipitantsinhigherconcentrations.Initially,thedropletofproteinsolutioncontainsaninsufficientconcentrationofprecipitantforcrystallization,butaswatervaporizesfromthedropandtransferstothereservoir,theprecipitantconcentrationincreasestoaleveloptimalforcrystallization.Sincethesystemisinequilibrium,theseoptimumconditionsaremaintaineduntilthecrystallizationiscomplete(Rhodes,1993;McRee,1993).

Simplyput,thehangingdropmethoddiffersfromthesittingdropmethodintheverticalorientationoftheproteinsolutiondropwithinthesystem.Itisimportanttomentionthatbothmethodsrequireaclosedsystem,thatis,thesystemmustbesealedofffromtheoutsideusinganairtightcontainerorhigh-vacuumgreasebetweenglasssurfaces.Figures3and4depictthehangingdropandsittingdropsystems,respectively(Rhodes,1993;McRee,1993).

Figure3.Diagramofhangingdropmethod.Reservoirsolution(blue)usuallycontainsbufferandprecipitant.Proteinsolution(red)containsthesamecompounds,butinlowerconcentrations.Theproteinsolutionmayalsocontaintracemetalsorionsnecessaryforprecipitationofparticularproteins.Forinstance,insulinisknowntorequiretraceamountsofzincforcrystallization(McRee,1993).

Figure4.Diagramofsittingdropmethod.Inthismethod,theproteindropsitsonapedestalabovethereservoirsolution,asopposedtohanging.

HighThrough-PutMethods:

Oneunavoidableaspectofcrystallizingaproteinfromscratchistheneedforalargenumberofexperimentsexploringthevariousconditionsthatarenecessaryforsuccessfulcrystalgrowth.However,itistedioustosetupandscreensomanydifferentexperimentalconditions.Thus,highthrough-putmethodsexisttohelpstreamlinethisprocess.Therearenumerouskitsavailabletoorderwhichapplypreassembledingredientsinsystemsguaranteedtoproducesuccessfulcrystallization.Usingsuchakit,ascientistavoidsthehassleofpurifyingaproteinanddeterminingtheappropriatecrystallizationconditions.Anothermethodthathasprovenusefulinsettingupavastnumberofcrystallizationsinvolvesrobotics.Whatwouldotherwisebeslowanderror-pronewhencarriedoutbyahumancanbeaccomplishedefficientlyandaccuratelywithanautomatedsystem.Clickheretoseeamovieofacrystallizationrobotinaction(http://www.syrrx.com/technology/index.htm).Roboticcrystallizationsystemsusethesamecomponentsdescribedabove,butcarryouteachstepoftheprocedurequicklyandwithalargenumberofreplicates.Eachexperimentutilizestinyamountsofsolution,andtheadvantageofthesmallersizeistwo-fold:thesmallersamplesizesnotonlycut-downonexpenditureofpurifiedprotein,butsmalleramountsofsolutionleadtoquickercrystallizations.Eachexperimentismonitoredbyacamerawhichdetectscrystalgrowth(Eurekalert,2000).

Links

Aphotogalleryofproteincrystals.

NASA"sproteincrystalgrowthhomepage.(Amicrogravityenvironmentisapparentlyconducivetosuccessfulcrystalgrowth.)

ProteinCrystallizationandDumbLuck.(AnessayonthehaphazardsideofproteincrystallizationbyBobCudney.)

**Unlessotherwisenoted,allfigurescreatedbyJohnM.Kogoy.

References

Berman,HelenM.,etal."TheProteinDataBank."NucleicAcidsResearch.28:235-242(2000).<http://nar.oupjournals.org/cgi/content/abstract/28/1/235>

Branden,CarlandJohnTooze.IntroductiontoProteinStructure.NewYork:Garland,1999(pp.374-376).

"TheCrystalRobot."Eurekalert.Accessed:February18,2003.<http://www.eurekalert.org/features/doe/2000-12/drnl-tcr061902.php>

McRee,DuncanE.PracticalProteinCrystallography.SanDiego:AcademicPress,1993(pp.1-23).

ProteinDatabank.Lastupdated:2003.Accessed:February17,2003.<http://www.pdb.org>

Rhodes,Gale.CrystallographyMadeCrystalClear.SanDiego:AcademicPress,1993(pp.8-10,29-38).

Tulinsky,A."TheProteinStructureProject,1950-1959:FirstConcertedEffortOfaProteinStructureDeterminationIntheU.S."TheRigakuJournal.16(1999).<http://www.rigakumsc.com/journal/Vol16.1.1999/tulinsky.pdf>

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