Table Of ContentDERIVATIZATIONOFRIBONUCLEASEA:ANHPLCANDMASSSPECTROMETRIC
ANALYSISOFMODIFICATIONBYCHEMICALANDMUTAGENICMETHODS
By
TRISHAJOYMtzNDEZ
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1999
AKNOWLEDGEMENTS
MytimespentattheUniversityofFloridawillalwaysrepresentformearather
remarkabletimeinmylife. Manypeoplehavecontributedtomakingthetimevaluable,
notonlywithregardtothewell-roundededucationIhavereceivedbutalsothe
memoriesthathavebeencreated. Iwishtoaddressthosewhohaveputagreatdealof
effortintomyendeavors.
Noeducationispossiblewithoutanenvironmentinwhichtoflourish. Therefore,I
wouldliketothanktheestablishment,theUniversityofFlorida,theGraduateSchool,
CollegeofLiberalArtsandSciencesandtheDepartmentofChemistryforallowingmeto
joinandleamwithinthisinstitution. SpecificallyIacknowledgethestipendandtuition
waiversIreceivedaswellastravelfunds,andIamgratefulforthisfinancialsupport.
Iwishtothankallthosewhohavebeenmycolleaguesinonecapacityor
another. Ishallstartwiththosehaveworkedwithmedayinanddayout,theRichardson
researchgrouppastandpresent. Ithasbeenapleasuretoworkwithandgettoknow
KenWeakley,CelesteRegino,DeonBennett,LindaNichols,HuirongYao,ChengXu,
JerroldMillerandMattRyan.Eachpersonhasenlivenedandaddedanimportant
dimensiontothefriendlyatmosphereoftheRichardsonresearchlabs. Iwillmissthese
friends. IspecificallyacknowledgeCelesteReginoforthesynthesisoftheDPAligands
andforaidinthecouplingreactions,andIamgratefultoLindaNicholsforthe
acquisitionofallESI/FTICR-MSdatapresentedhere.
Iwishtothankthosepeopleinthemassspectrometryserviceslabwhohave
becomerespectedcolleagues:Dr.DavidPowellandDr.JodieJohnson. IthankDr.
a
PowellfortheacquisitionoftheMALDI/TOF-MSdatapresentedinthisdissertation,his
muchappreciatededitingskillsforthepreparationofthismanuscriptaswellashis
financialsupport. IamgratefultoJodiefortheacquisitionofallHPLC/ESI/QIT-MSdata
presented. IalsothankJodieforhisgreatideas,smilingface,qualityofcharacterand
abilitytolistenthathavemadeourfriendshipofhighestvaluetome. Ialsomustthank
Dr.NancyDenslowfortheuseoftheMALDI/TOF-MSintheICBRProteinCoreandfor
heradviceinproteinchemistry.
IwanttothankthepeopleintheBennergroupwhohavecontributedtomy
effortsindifferentways. LeeRaleyhasperformedasignificantamountofworkforthe
creationofthemutantRNaseAandtaughtmewhatIknowaboutproteinexpression.
StefanLutzhasbeenacolleaguebeyondcomparefromtheverybeginning. From
classestoqualifyingexamstopracticingforpresentations,ithasbeenmygoodfortune
tohaveStefanasmyclassmateandfriend. Dr.StevenBennerhasbeengenerousin
hissharingofequipmentformyresearchandenlighteningasateacher,oneIhopeto
neverforget.
Icannotforgetthepeoplewhomakethebureaucracyofschoolmucheasierto
navigate:allthoseinthechemistryfiscaloffice,thegraduatecoordinator’sofficeandthe
inorganicdivisionaloffices,especiallyAmandaGarriguesandMaribelLisk.
MytimeatUFwouldneverhavebeenasenjoyableasitwasifnotforthe
friendshipsofmany,includingPerlaSklar,JuliaRusak,LindaNichols,andmost
importantlyJenniferTonzello. Thesewomenwillbelifelongfriends.
Mydeepappreciationgoestomyresearchadvisor,ProfessorDavidRichardson.
Davehashelpedmebecomeabetterresearcher,improvemywritingskillsandleam
howtothickenmyskin,allskillsthatwillimprovemyprofessionalfuture. Theareasin
whichandthesheervolumeofinformationIlearnedfromDavecannotbedescribed
here. LetitbesufficientthatIsayIamtrulygratefulforhiseffortsasanadvisor.
Finally,myheartfeltthanksgoestomyimmediatefamily,whoseloveformehas
madepossibleeverythinggoodinmylife. Mymother,Lora,hasbeenmybiggest
cheerleaderandmosthelpfulfriend. Hercourageandworkethichavebeenthebest
exampleofawomantobeemulated. Myson,Alexander,hasmademeamoreefficient
student,butmoreimportantlyhispresencehastaughtmegratitudeforthingsmost
importantinlife. Myhusband,Edras,hasbeenmypartnerandfriendforlongerthanI
canremember. Hiscontributionstomylifeseeminseparablefrommyown,andthe
workpresentedhereisjustoneofmanyendeavorsthatwouldnotbepossiblewithout
hisunconditionallove. MostimportantlyIthankthegoodLordforHisabundantgrace.
IV
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS
ABSTRACT
3
CHAPTERS
1 INTRODUCTION
4
ChemicalModificationofProteins
ElectrosprayMassSpectrometry
ESI-MSApplicationstoProteinCharacterization
ESI-MSforAnalysisofChemicalModificationsandBioconjugation
5 inProteins
2 CHEMICALMODIFICATIONOFRIBONUCLEASEA
Introduction 30
ExperimentalProcedures 36
ResultsandDiscussion... 41
Conclusions 66
THEMAPPINGOFSMPT-MODIFIEDRNASEAI:PROTEOLYTICDIGESTION
WITHTRYPSINANDESI/FTICR-MSANALYSIS 69
Introduction
ExperimentalProcedures 74
ResultsandDiscussion 77
Conclusions ®7
THEMAPPINGOFSMPT-MODIFIEDRNASEAII:PROTEOLYTICDIGESTION
WITHSUBTILISINANDHPLC/ESI/QIT-MSANDMSnANALYSIS 88
Introduction
ExperimentalProcedures 9^
CReosnuclltussiaonndsDiscussion ^933
BIOCONJUGATIONOFRNASEATOASYNTHETICCOFACTOR:
PRELIMINARYWORK
v
9
Introduction 114
ExperimentalProcedures 11
ResultsandDiscussion 123
Conclusions 129
6 GENERALCONCLUSIONS 132
APPENDIX 138
LISTOFREFERENCES 144
BIOGRAPHICALSKETCH 160
vi
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
DERIVATIZATIONOFRIBONUCLEASEA:ANHPLCANDMASSSPECTROMETRIC
ANALYSISOFMODIFICATIONBYCHEMICALANDMUTAGENICMETHODS
By
TrishaJoyM6ndez
December1999
Chairman:ProfessorDavidE.Richardson
MajorDepartment:Chemistry
Chemicalmodificationofproteinsisademonstratedmethodforincorporating
newligandsintoanenzymethroughthereactionofresiduesidechainswithvarious
chemicalreagents. Reactionofanenzymeresiduewithamodifyingreagentthat
incorporatesaligandofinteresttothesystemresultsinpotentialnovelchemicalactivity
oftheenzyme,creatingabioconjugate. Abioanalyticalmethodforthedevelopmentand
characterizationofabaseproteinsystemtobeusedasabioconjugateispresented.
ThemodificationofribonucleaseAwiththeheterobifunctionalcross-linker,4-
succinimdyloxycarbonyl-methyl-a-[2-pyridyldithio]-toluene(SMPT),isdescribed. RNase
Ahas11sitesofpotentialmodificationbytheSMPTreagent. Trackingthe2-
dimensionalseparationandproteolyticdigestionofSMPT-modifiedRNaseAwith
ESI/FTICR-MSandHPLC/ESI/QIT-MSdemonstratesthedetailedinformationaboutthe
numberofSMPTmodificationsandsitesofmodificationthatcanbeobtainedby
applicationofthesetechniques.AnalysisofnativeandmodifiedRNaseAtrypticdigests
byESI/FTICR-MSresultedintheidentificationofthesitesofmodification. Semi-
vii
quantitativeresultsofthereactivityofcertainlysineresiduestowardthecouplingreagent
SMPTarepossible.Twosites(lysines1and37)arehighlyreactive,whilethreesites
(lysines41,61and104)appeartobeunreactiveundertheconditionsused. Digestion
ofthemodifiedandunmodifiedRNaseAbysubtilisinfollowedbyexaminationwith
HPLC/ESI/QIT-MSresultedinthequantificationofthepropensityofsubtilisintocleave
RNaseAandSMPT-modifiedRNaseAatvariouspeptidelinkages,whichhasnotbeen
previouslydemonstrated. HPLC/ESI/QIT-MSandMS"enabledfurtherinvestigationof
modificationonlysines1and7,includingmodificationatthes-anda-aminopositionson
lysine1. CouplingofsinglymodifiedRNaseAtoasyntheticcofactorofinterest,2,2’-
dipyridylamine,isdemonstratedasanexampleoftheassemblyofthebioconjugate.
ThecomplementtochemicalmodificationofRNaseAforthedevelopmentofa
systemforbioconjugationissite-directedmutagenesis. Preliminaryworkonthe
characterizationandcouplingofthemutatedRNaseA(F8C)usingMALDI/TOF-MSis
presented.
viii
CHAPTER1
INTRODUCTION
ChemicalModificationofProteins
Thechemicalmodificationofproteinsisausefultoolfortheelucidationof
informationmostbasictoenzymechemistry,thatis,theunderstandingofsubstrate
bindingandsubsequentbimolecularcatalysis. Therationaldesignofchemicalmodifiers
hasenabledstructuralinformationaboutproteinsandenzymestobeobtainedpriorto
themodemtechniquesofX-raycrystallographyandnuclearmagneticresonance(NMR)
spectrometry. Byintroducingnewchemicalgroupsintoaproteinviacovalent
interaction,informationcanbelearnedaboutthechemicalnatureandrelativereactivities
oftheaminoacidsthatarepresent. Residuesmostsusceptibletochemicalmodification
arelysineandarginineattheirnucleophilice-animogroups,tyrosineatitshydroxy
group,cysteineatitssulfhydrylgrouporcross-linkeddisulfide,histidineatitsimidazole
group,tryptophanatitsindolegroup,glutamicacidandasparticacidattheircarboxylic
acids,theN-terminalamino,andC-terminalcarboxylgroups. Duetothedifferencesin
thechemicalnatureofthesegroups,theirreactivitiesvarywithdifferentchemical
modifiers,changingpH,andbuffercomposition(1-3). Exploitationofthesechemical
differencesthroughchemicalmodificationhasresultedinthousandsofpublications
demonstratingstructuralandfunctionalinformationforproteins. Anoverviewof
researchexamplesispresentedhereillustratingnotonlytheevolutionofchemical
modificationasastandardtoolfortheinvestigationofproteinsbutalsocreative
expansionstothebasicchemicalmodificationsfortheaccomplishmentofinnovative
researchgoals.
1
2
Oneofthefirstcomprehensivechemicalmodificationstudieswasdemonstrated
in1948,byFraenkel-Conratetal.(4). Theseresearchersinvestigatedtheprotease
trypsin,itsinhibitor,ovomucoidandthemodeofinteractionbetweenthetwothrough
chemicalmodification. Aseriesofchemicalderivatizationswereperformedontrypsin
andovomucoidincludingacetylation,esterification,epoxidation,reduction,couplingwith
diazobenzenesulfonicacid,iodination,andsoon. Suchmodificationsresultedinthe
determinationthattrypsininteractswithasubstratethroughgroupsotherthanitsamino
groupsbutcombinesthroughitsaminogroupswiththeacidgroupsoftheinhibitor,
ovomucoid. Astabilizationrolewasassignedtotheotherimportantresiduesintrypsin.
Fraenkel-Conratetal.(5)usedchemicalmodificationagainin1950toprobethe
iron-bindingcapabilityofconalbumin,anenzymefoundineggwhites. Itwasreported
thatallchemicalmodificationsperformedinthisresearch,includingacetylation,
iodinationorcouplingofphenolicacidandimidazolegroups,substitutionofamideof
guanidylgroupsanddisulfidereduction,resultedininactivationofiron-bindingcapability,
demonstratingthecomplexityoftheinteractionsrequiredformetal-binding. Analytical
techniquesavailableatthattimedidnotallowfurtherinformationtobeextractedfrom
thechemicalmodificationresults,despitetheunderstandingofthechemistryinvolvedin
themodifications.
In1962,ageneralmethodforcovalentmodificationofactivesiteresidueswas
presentedbyWofsyeta/.(6). Eventhoughthespecificexampleusedtodemonstrate
thetheorywasanantibody,itsdirectapplicationtoenzymeswasclear.Themethod,
termedaffinitylabeling,madeuseofamodificationreagentthatisdesignedtohavean
affinityforaspecificsiteontheprotein. Onceboundbynon-covalentinteractions,the
reagentandproteinundergoachemicalreactiontocovalentlybindthereagentorpartof
thereagenttotheproteinintheselectedsite. However,itwasnotedthat“...conclusive
proofthatlabelingoftheactivesitehadoccurredcouldonlybeachievedthroughthe
