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OverviewofCrosslinkingandProteinModification
Anumberoftechniquesforstudyingthestructureandinteractionofproteins,aswellasformanipulatingproteinsforusein
affinitypurificationordetectionprocedures,dependonmethodsforchemicallycrosslinking,modifyingorlabelingproteins.
Crosslinkingistheprocessofchemicallyjoiningtwoormoremoleculesbyacovalentbond.Modificationinvolvesattachingor
cleavingchemicalgroupstoalterthesolubilityorotherpropertiesoftheoriginalmolecule."Labeling"generallyreferstoany
formofcrosslinkingormodificationwhosepurposeistoattachachemicalgroup(e.g.,afluorescentmolecule)toaidin
detectionandisdescribedinotherarticlesintheProteinMethodsLibrary.
Theentiresetofcrosslinkingandmodificationmethodsforusewithproteinsandotherbiomoleculesinbiologicalresearchis
oftencalled"bioconjugation"or"bioconjugate"technology.(Conjugationisasynonymforcrosslinking.)
Proteinstructure
Covalentmodificationandcrosslinkingofproteinsdependsontheavailabilityofparticularchemicalsthatarecapableofreactingwiththespecifickindsoffunctionalgroupsthatexistinproteins.In
addition,proteinfunctionandstructureareeitherthedirectfocusofstudyortheymustbepreservedifamodifiedproteinistobeusefulinatechnique.Therefore,thecompositionandstructureof
proteins,andthepotentialeffectsofmodificationreagentsonproteinstructureandfunction,mustbeconsidered.
Proteinshavefourlevelsofstructure.Thesequenceofitsaminoacidsistheprimarystructure.Thissequenceisalwayswrittenfromtheaminoend(Nterminus)tothecarboxylend(Cterminus).
Proteinsecondarystructurereferstocommonrepeatingelementspresentinproteins.Therearetwobasiccomponentsofsecondarystructure:thealphahelixandthebetapleatedsheet.Alpha
helicesaretight,corkscrewshapedstructuresformedbysinglepolypeptidechains.Betapleatedsheetsareeitherparallelorantiparallelarrangementsofpolypeptidestrandsstabilizedbyhydrogen
bondsbetweenadjacentNHandCOgroups.Parallelbetasheetshaveadjacentstrandsthatruninthesamedirection(i.e.,Ntermininexttoeachother),whileantiparallelbetasheetshave
adjacentstrandsthatruninoppositedirections(i.e.,NterminusofonestrandarrangedtowardtheCterminusofadjacentstrand).Abetapleatedsheetmaycontaintwotofiveparallelorantiparallel
strands.
Tertiarystructureisthefullthreedimensional,foldedstructureofthepolypeptidechainandisdependentonthesuiteofspontaneousandthermodynamicallystableinteractionsbetweentheamino
acidsidechains.Disulfidebondpatterns,aswellasionicandhydrophobicinteractionsgreatlyimpacttertiarystructure.Quaternarystructurereferstothespatialarrangementoftwoormore
polypeptidechains.Thisstructuremaybeamonomer,dimer,trimer,etc.Thepolypeptidechainscomposingthequaternarystructureofaproteinmaybeidentical(e.g.,homodimer)ordifferent(e.g.,
heterodimer).
Thefourlevelsofproteinstructure.Thesequenceofaminoacids,representedbybluedots,joinedbypeptidebonds,comprisetheprimarystructure.Thepropertiesoftheconstituentaminoacids,inthecontextofthecellular
environment,largelydeterminespontaneousformationofthehigherlevelstructurethatisessentialforproteinfunction.
CrosslinkingReagentsTechnicalHandbook
This45pageguideisofvaluetothenoviceaswellasthosewhohavepreviousexperiencewithcrosslinkingreagents.Itbeginswithabasic
discussiononcrosslinkingandthereagentsthatareused.Theguidealsocontainsadiscussiononvariousapplicationswherecrosslinkinghas
beenapplied,includingthepowerfullabeltransfertechniqueforidentifyingorconfirmingproteininteractions.Crosslinkingchemistryisaddressed
inaneasytofollowformatdesignedtoconveytheimportantinformationyouneedwithoutgettinglostindetails.EachPiercecrosslinkingreagent
isshownalongwithit'sstructure,molecularweight,spacerarmlengthandchemicalreactivity.Thehandbookconcludeswithalistofexcellent
referencesoncrosslinkeruseandaglossaryofcommoncrosslinkingterms.
CrosslinkingReagentsTechnicalHandbook
Thecompletestructureofafunctioningproteininvolvesmorethanpolypeptidechainsatthefourlevelsofstructure.Variouscovalentmodificationsoftenoccur,eitherduringorafterassemblyofthe
polypeptidechain.Mostproteinsundergocoand/orposttranslationalmodifications.Examplesincludephosphorylation(ofserine,threonineortyrosineresidues),glycosylation,andubiquination.
Knowledgeofthesenativemodificationsisextremelyimportantbecausetheymayalterphysicalandchemicalproperties,folding,conformationdistribution,stability,activity,andconsequently,function
oftheproteins.Thestudyofposttranslationalmodification(adifferentmeaningfromtheproteinmodificationbeingdiscussedinthepresentarticle)isanimportantareaofresearchseerelated
articlesforadiscussionofthattopic.
Becausethestructureofaproteindictatesitsbiologicalactivity,characterizationofproteinstructurecontinuestobeanimportantareaofresearch.Proteinsarerelativelyeasymoleculesto
manipulate,andproteincrosslinkingandchemicalmodificationmethodsarecommonlyusedtodeterminetherolesofindividualaminoacidsidechainsinthephysical,chemical,andbiological
propertiesofproteins.Furthermore,oncetheirbiologicalpropertiesareunderstood,proteinscanoftenbeusedtostudyothersystemsinapplicationssuchaspreparingantibodyenzymeconjugates
forimmunoassays.
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Posttranslationalmodification
Overviewofproteinproteininteractionanalysis
Functionaltargetsandreactivegroups
Despitethecomplexityofproteinstructure,includingcompositionwith20differentaminoacids,onlyasmallnumberofproteinfunctionalgroupscompriseselectabletargetsforpracticalbioconjugation
methods.Infact,justfourproteinchemicaltargetsaccountforthevastmajorityofcrosslinkingandchemicalmodificationtechniques:
Primaryamines(NH2):ThisgroupexistsattheNterminusofeachpolypeptidechainandinthesidechainoflysine(Lys,K)residues.
Carboxyls(COOH):ThisgroupexistsattheCterminusofeachpolypeptidechainandinthesidechainsofasparticacid(Asp,D)andglutamicacid(Glu,E).
Sulfhydryls(SH):Thisgroupexistsinthesidechainofcysteine(Cys,C).Often,aspartofaprotein'ssecondaryortertiarystructure,cysteinesarejoinedtogetherbetweentheirsidechains
viadisulfidebonds(SS).
Carbonyls(CHO):Thesealdehydegroupscanbecreatedbyoxidizingcarbohydrategroupsinglycoproteins.
Foreachoftheseproteinfunctionalgrouptargets,thereexistonetoseveraltypesofreactivegroupsthatarecapableoftargetingthemandhavebeenusedasthebasisforsynthesizingcrosslinking
andmodificationreagents.
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Chemistryofcrosslinking
Crosslinkingproteins
Crosslinkingistheprocessofchemicallyjoiningtwoormoremoleculesbyacovalentbond.Crosslinkingreagents(orcrosslinkers)aremoleculesthatcontaintwoormorereactiveendscapableof
chemicallyattachingtospecificfunctionalgroups(primaryamines,sulfhydryls,etc.)onproteinsorothermolecules.
Attachmentbetweentwogroupsonasingleproteinresultsinintramolecularcrosslinksthatstabilizetheproteintertiaryorquaternarystructure.Attachmentbetweengroupsontwodifferentproteins
resultsinintermolecularcrosslinksthatstabilizeaproteinproteininteraction.Alternatively,ifthesamplewereamixtureoftwopurifiedproteins(e.g.,anantibodyandanenzyme),theintermolecular
crosslinkcreatesaspecificconjugateforuseindetectionprocedures.Finally,attachmentbetweenaproteinandachemicalgrouponasolidmaterial,suchasaglassslideorbeadedresin,resultsin
immobilizationoftheproteintothesurfaceproteinimmobilizationisthebasisformanykindsofassayandaffinitypurificationsystems.
Thus,crosslinkingisusedformanypurposes,includingto:
Stabilizeproteintertiaryandquaternarystructureforanalysis.
Captureandidentifyunknownproteininteractorsorinteractiondomains.
Conjugateanenzymeortagtoanantibodyorotherpurifiedprotein.
Immobilizeantibodiesorotherproteinsforassaysoraffinitypurification.
Attachpeptidestolarger"carrier"proteinstofacilitatehandling/storage.
Crosslinkersareselectedonthebasisoftheirchemicalreactivities(i.e.,specificityforparticularfunctiongroups)andotherchemicalpropertiesthatfacilitatetheiruseindifferentspecificapplications:
Chemicalspecificity,includingwhetherthereagenthasthesameordifferentreactivegroupsateitherend(i.e,doesithaveahomobifunctionalorheterobifunctionalstructure?)
Spacerarmlength,includingwhetherthearmiscleavable(i.e.,canthelinkagebereversedorbrokenwhendesired?)
Watersolubilityandcellmembranepermeability(i.e.,canthereagentbeexpectedtopermeateintocellsand/orcrosslinkhydrophobicproteinswithinmembranes?)
Spontaneouslyreactiveorphotoreactivegroups(i.e.,willthereagentreactassoonasitisaddedtoasampleorcanitsreactionbeactivatedataspecifictime?)
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Chemistryofcrosslinking
Crosslinkersataglance(tableofallcrosslinkers)
Crosslinkingapplications
Crosslinkerselectionguide(interactiveguide)
Crosslinkingproteininteractionanalysis
Chemicalmodificationofproteins
Proteinanalysisanddetectiontechniquesoftenrequiremorethandirectconjugationwithabifunctionalcrosslinkeroractivatedlabelingreagent.Forexample,inmanysituations,specializedprotein
modificationsareneededtoaddmolecularmass,increasesolubilityforstorage,orcreateanewfunctionalgroupthatcanbetargetedinasubsequentreactionstep.
Simplystated,proteinmodificationreagentsarechemicalsthatblock,add,changeorextendthemolecularreachoffunctionalgroups.(Inamoregeneralsense,proteinmodificationalsoincludes
proteasesandreducingagentsforcleavingpolypeptides,butthosearedistincttopicsthatarebetterdiscussedinotherarticles.)Threeexamplesaresufficienttodescribethetypesandpurposesof
modificationreagents:
Pegylation:Chemicallyattachingsingleorbranchedchainpolyethyleneglycol(PEG)groupstoproteinsisaformoflabelingormodificationthatisprimarilyusedtoconferwatersolubility
and/orinertmolecularmasstoproteins.FormsofPEGthathavebeensynthesizedtocontainreactivechemicalgroupscomprisereadytouse,activatedreagentsforpegylation.
Blocksulfhydryls:Proteinsulfhydryls(sidechainofcysteine)areimportantregulatorsofproteinstructureandfunction.Certainreagentsarecapableofreactingpermanentlyorreversiblywith
sulfhydrylgroups(e.g.,NEMorMMTS,respectively).Thesereagentsaddaverysmall"cap"onthenativesulfhydryl,enablingtheactivityofcertainenzymestobecontrolledforspecificassay
purposes.
Convertaminestosulfhydryls:SATAandrelatedreagentscontainanaminereactivegroupandaprotectedsulfhydrylgroup.Byreactthecompoundtoapurifiedprotein,thesidechainof
lysineresiduescanbemodifiedtocontainasulfhydrylgroupfortargetingwithsulfhydrylspecificcrosslinkersorimmobilizationchemistries.Themethoddoesnotactuallyconverttheamineinto
asulfhydrylratheritattachesasulfhydrylcontaininggrouptotheprimaryamine.Theeffectisalsotoextendthelengthofthesidechainbyseveralangstroms.
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Modificationreagentsforaminoacidsidechains
Reducingagents
Pegylationreagents
Proteases
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Polyethyleneglycol(PEG)reagentsandpegylation
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