Production and Structural Studies of Lung Surfactant Protein B (SP-B) Peptides
MahzadSharifahmadian July 2011,MemorialUniversityof Newfoundland
Submitted in partialfulfillmentofthe requirements fortheDegree ofMaster
Departmentofl3 iochcmistry Memorial Univers ityof Newfound land
St.John's ,NLAIB3X9
Lung surfactant is amixture of lipids and proteinswhich iscritica I for normalbreath ingbyliningthe air-wa ter interfacetoreduc ethe surface tension.Lung surfactant proteinB(SP-B) istheonlyessentia l prote in component oflung surfactantcomplex duetothe lethalit yof any SP-B deficiency. Itisthou ghtthatSP-Bfuncti on sbyenhancin glipid rearrangementsat vari ou sphases of the breathin g cycle.However,thehigh- resoluti on struc tureand mechan ism ofSP-Barenotyet understood.In the firstpart ofthisresearch ,SP-Banda 7- residu edeleti onmutant of SP-B wereproduc ed reco mbinantlyand partia llyc haracterized. In the second part ofthis rese arch,circular dichroism , solution andsolid-state nuclear magneticresonance (NMR) meth odswereusedtoassessthestructureof twoSP-B-basedpeptid es, Supe r-M ini-BandN-termina l insertion seq uence (SP-BI_7).Super-M ini-B is compose dofthe N-terminal7-res id ue inserti on sequenceand the N-andC-terminal helic es ofSP-B. Interestin gly,it was obse rved that Super-Mini-B produces greater lipid memb raneperturbation than the pept idewhichlacksthe N-term inal inserti on seq uence(i.e.Mini- B).Comparing theresult s of structura lstudieson Mini-B,SP-BI_7and Supe r-Mini-B help sunveilthe contribution of the7-residu einsert ion sequence tothe functionof SP-B.
Acknowledgements
Iwouldfirst like tothankmysupervisor,Dr.ValerieBoothfor her wonderfulguidance andsupport.I would also liketothank Donn aJ ackrnan, Dr. Muzaddid Sarker,Dr.MichaelHayley and DharamarajuPalleboinawho helpedme greatly throughoutmyproject.Iacknowledge thesupportive and encouragingrolesof my committee membersDr.MikeMorrow and Dr.
Kaushik Nagthroughoutthewholeprogram .Iwould like tothankDr.
DavidHeeley,Craig Skinner,Marie Codner, for theirhelp(Biochemi stry Department,Memorial University,NL,Canada),as well Dr.Alan Waring and hiscolleaguespreparin g oneof thepeptid e samples usedinthiswork (DepartmentofMedicine,UCLASchool of Medicine,CA, USA).The pan- university Core Research Equipmentand Instrum entTrainin g Network (CREAIT)at Memorial University forfacilitatingsomeNMR experiments and performin gDNA sequencingexperiments. Iamalsogratefulforthe fundingobtainedfor thisproje ctfrom Canadian Institute sof Health Research (CIHR) tomysupervisor.
Iwouldalso like tothank all mylovelyfriends and people who madeIiving inSt.John'slikebeing at home.
Acknowledge ments
Llstof Figur es
Chaplc r l: Introduction
Chaplcr 2: Ma ter ials and Mclhods 2.I. SelJuenceidentificalionof SI' -B encod cdplasmid 2.2.Tra nsfor ma tionof theplasmid10the express ion cell line 2.3.Recombinanlcxpr cssionoffulllength surfacta nt proteinB 2.4. Prod uclion & reco mhinanlc xprcss ionofl heN- le r mina l
2.5.ldent ificat ion of theexpress cd peptid e
2.6.Che mica lsynt hesisand purificali on of N-Ier mina l fragment of SP-B(S P-B,_7)
2.7.So lulion nuciea r mag nelic rcso na ncc(N MR) 2.8.So lid-sla le nuciear null:nelic res ona ncc(NM R)
2.9.Cir cu la r dichroism(CD ) spcctroscop y
Cha pter 3:Result s
3.1. Detectionofexp resse d rccomhinant full length SP-B 3.2.ln\'cstigatingdisruptionofprotcin synth csis andproteol)'sis
inducedh)'str ess-acti\'atedprotcin s 3.3.In\'cstigating effectofcclldisruptiontechnique son the
exp ress ed protein 3.4.Optimizing growth conditions
3.5.I'roductionofano\'cI SP-Hpcptidc; N-terminaldeletcd SP-B pcptidc
3.6.ldentiflcationofexp ressed rccomhinantN-tcrminaldeletcdSI'-H 3.7.Productionand characterization ofN -tcr mina l insertion scqucnce
3.8.Solution nuclearmagneticrcson an ce sp ectr oscop y (NMR ) prcliminary structuralcharacterizationof N-terminalinsertion peptid e
3.9.Ch ar acteri zin gthe structure of Sup cr-l\Iini-B 3. I O.S olu tio n nucica r ma gnetic rcso na nces pectr oscopy(NM R)
st r uct u r a l cha r a cter iza tion ofS upcr- Mi n i-B
3.II.Solidsta te nuclearmagneticresonan ce (NMR)charac tc rizationof lipid-protein intera ction ofSuper -Mini-B
Chapter 4:Discussion
Supplementa lmateria l
TableI. Amino acidsequencesof S P-B. Mini-Band Super-Mini-B Table2. Thes equences offo rwarda nd reverse primers designed fors ite 37
Table3.MS/MSanalysis confirmedthepresence oftwo fragmentsfrom
Table-t.OptimizingIPTG concentrationtomaximize protein expression.
Table5. MS/MSanalysis ofSN-SP-B trypsindigested sam ple identified two fragmentswith theexpectedsequence
Table6.Secondarystructuralcontentof2mMSP-BI_7inthepresence 65 of300mM SOS NMRbufferand40%HFIPatpH 5 and 25°C.
Tahle7.IHChemicalshiftsof the N-terminalinsertionsequenceofSP-B from theTOCSYexperiment on SP-BI_7
Table8.Secondarystructuralcontentof I mM Super-Mini-B.in the presence of150 mMSOS(plus 90%H20 and 10%020)aswellas in 40%HFIP(plus 50%H20and 10%020)at pH 5.
Ta ble9.IHChemicalshiftsofthe N-terminalinsertion sequence ofSP-B fromtheTOCSYexperimenton Super-Mini-B
List of Figures
Figure \.Schematic diagramofthehumanrespiratory system Figure2. Schematicdiagram of matureSP-B and SP-B homodimer Figure3. Amodel representing roleo f SP-B duringco mpression
(a.pointing inarrows)andexpansion (a.pointingoutarrows) of breathin g cycle.
Figure 4.SchematicdiagramofSP-B peptidessynthe sizedbasedon thepredictedhelicalsegments ofSP-B.
FigureS .Arterial oxygenation and dynamiccompliance in ratssubjected to removaloflungsurfact antby invivo lavage.
Figure6. Thehigh-resolution structures ofthreet errninalfragment s of SP-B determinedby solutionNMR.
Figure7.Westernblot analysisof thecell extract harvestedfrom the overnightculture ofcellsencoding SN-SP-Bplasmid Figure8.MS-MSspectra ofSN-SP-B sample produced usingMALDI-TOF
mass spectroscopy.
Figure9.Westernblotrepresentationof time course colle cted cultures
Figu re 10. Westernblot analysisof100 mlcellextractssubjec ted to differentcelldisruption techn iques
Figure I\. The expectedaminoacidsequence ofN -terrninal deleted SP-Bpeptid e
Figure12. Westernblot analysis ofthecell extract froman overni ght induced cultureencodingSN-Nd-SP-B
Figure 13. MS/MS spectra resultin gfromMALDI-TOF mass spectrosc opy ofS N-Nd-SP-Btrypsindigested sample
Figu re l 4.HPLC spectrumof SP-BI_7eluted with an increasing acetonitrile gradient
Figure 15.Far-UV COspectrum of2mMN-terminal SP-Bpeptidedissolved in 40%HFIP(plus 50%H20and 10%020)at pH 5 (red)aswell asin the presenceof300 mM SOSNMR buffer containing90% H20 , 10%020,0.2mM OSS,and300 mM deuteratedSOS (green).
Figure 16. 20 TOCSY spectra of2mMSP-BI_7in300 mM SOS,at pH 5 69,70 and 45°C,panel shows the HN-H Xcorrelationregionand(B)
atpH5 and 45°C afteradditionof0.6mM16-0 SA.
Figu re 17.Effectof 0.6mM 16-0SAonN-terminalinsertionpeptideof SP-B Figu re 18.Far-UVCOspectrum of I mM Super-Mini-B dissolvedin40%HFIP 73
(plus 50% H20and10%020)at pH5 aswellasin the presenceof 150mMSOS(90% H20 ,10%020 ,0.2mM OSS,and 150 mM
deuter atedSOS).
Figu re 19.Far- UV COspectrumof1.5andImM Mini-B dissolvedin 90%H20,10%020 ,0.2mM OSS,andISOmM deuteratedSOS.
Figu re 20. IHNMRspectra ofSuper-Min i-Bweredissolvedat three 77,78
Figu re 21.IHNMRspectraof 0.9mM Super-Mini-B in 40%HFIP buffer 80,81 atvarying temperatures
Figure 22. IHNMRspectra of 1 mMSuper-Mini-B ina150 mM SOS 82,83 at varyingtemperatures
Figure 23.lHNMRspectraofMini-Bat pH 7and37°C,Super-Mini-Bat 84,85 pH 7 and37°Candat pH5 and 45°C
Figure 24. lHNMRspectraof1 mMSuper-Mini-B in 150mMSOS buffer 87,88
Figure 25. IHNMRspectraof 1.5 mM Mini-B at pH7 and3?OC (blue), 89.90 ImMSuper-Mini-Bat pH7 and 37°C(green)andatpH5 and 45°C (red).
Figure26. SolutionNMR20NOESYspectrumof 1 mMSuper-Mini-B dissolved ina150mM SOSat pH7 and
3ic
Figure 27.SolutionNMR20 NOESYspectrumof1 mMSuper-Mini-B dissolved 93 in a solutionof 90% H20 .10%020 , 0.2mM OSS,and 150mM deuteratedS OSat pH5 and 45°C.
Figure 28.Overlaidfeatureof2 0NOESY spectraof 1mMSuper-Mini-B 94,95 in 150mM SOSsolutionat pH7 and37'cand pH 5,45'c Figure 29.Overlaidfeatureof2 0NOESYspect raofSuper-Mini-B in 97,98
150 mM SOSsolutionat pH5 and450C and Mini-Bin 150mM SOSpH5 and37'c
Figure 30.Effectof0.6mM16-0 SA onN-termi nalresiduesof1 mM Super-Mini-B in 150mM SOS
Figure 31.20OOSY spectraof1 mMSuper-Mini-BinSOSat30'c Figure 32.211spectraof mechanically oriented POPC-d31:POPG 7:3 with
(upperpanel)andwithout(lowerpanel) 1 mol % Super-Mini-B.
Figure 33.2HNMR spectraofmech an icall y oriented bilayerscomp osed of POP C-d31:POP G (7:3,w/w)in thepresence ofSuper-Mi ni-B(1 mol%)
Figure 34. Orderparameter profilesOrde r parameterprofil es ofPO PC-d31:PO PG withand withoutSuper-Mi ni-B,Mini-Bandoverlaidfeature Figure 35.Echo Decay valueforPOP C-d31:POP G (7:3,w/w)in the abse nce
(A) andprese nce(B)of1 mol%Super-Mi ni-Bat35°C.
Figure SI.SDS-PAGEgel,Comassiesta inedofpurified SN-SP-B.
16-doxyl-stearicacid
acute respirato rydistress syndrome
5-bromo-4-chloro-3-indolyl-phosphate
cellpenetratingpeptide
dynamiclight scattering
dimethylformamid e
diffusionorderedspectrosco py
dipalmit oylphosphatidylcholine
sodium2,2-dimethyl-2-silapentane-5-sulfonate
ethylenediaminctetraaceticac id
9-tluorenylmethyloxycarbonyl
hexatlu oro isopropanol
high-pressureliquid chromatography
P-D- l -thioga lactopyranoside
MALDI-TOF MS matrix assistedlaser desorptionionization-time of flightmass spectrometry
nitrobluetetrazo lium
nuclear magneticresonance
nuciearoverhauser -etTectspectro scopy
neonatalacuterespiratory distress syndrome
phosphatidy lcholine
polymerasechain reaction
phosphat idyleglycerol
phcnylmcthancsulfonyltluoridc
Pope-dll deuterated I-palmitoy l-2-oleoyl-sn-glycero-3-phosphocho line
I-palmitoyl-2-oleoyl-sn-glycero-3-phospho-( I'-rac- glycerol)
partsper million
polyvinylidencfluor idc
sodium dodecylsulphate
SDS-PAGE sodium dodecyl sulfate polyacrylamidegelelectrop horesis
staphylococcus nuclease
surfactant protein A
surfactant protein B
SP· BI_7 N-terrninalinsertionsequence
surfactantproteinD
trisbufferedsaline
2,2,2-trifluoroe thanol
total correlationspectroscopy
Therespiratory system functions asthe supplier ofoxygentothe blood.Gasdiffusionhappens at the alveo lar level inverycloseproximityt o lung capillaries.Thereareabout300 million alveo li in thelungsproviding a total surfacearea ofabout 140 m1(Guytonand Hall, 2006).Thealveolar wallsare linedby alveolarepithelialcells which are in directcontactwith respiratorygases (Figure I).
Figure1.A schematic diagram ofthehuman respiratory system. Lung surfactant,alipid-protein complex,linestheinner surfaceof alveo liand redu ccsth e surfacet ension atthe air-w aterintcrfacet o facilitatebreathin g.
Theconstantexposure to environmentalgasesand particles requiresthepresence of aO .2mm thickwaterlayer coating the inner surface of the alveoli.This layerpreventsthedehydration (Bastackyetal. 1995).
Whenwaterformsa surface with air,water molecules stronglyattracteach other;the forcesof attraction betweenwater moleculesare strongerthant he
interface.Thesurfacetensionforcesairout causing thealveolito collapse andconseq uentlydisruptsrespiration(Guytonand Hall, 2006).The natural remedytodecreasethe surface tensionand maintainintlatio nis thepresence oflung surfactant, a complexmixture oflipids andproteins (Whitsett et al., 20 10). Lungsurfactant complexdecreasesthesurface tension by formation ofthesurface activefilms at the alveo lar levelandtransfer ofsurface active materia lsbetween the air-waterinterfaceand hypophases(Serranoetal., 2006).
Deficiencyorinact ivationoflung surfactant eitherby premature birth,genemutationorlunginjuryleadsto severerespiratoryd isorders.T he pathophysiologicaleffectof'surfactantdeficiencywas firstdiagnosedin premat ureinfantswithneonata lrespiratorydistresssyndrome(NRDS).
Lungsurfactantdoes not normallybegintobesecretedinto the alveoliuntil thesixth orseventhmonths of pregnancy,insome cases,even later than that. Thusmanypremature babieshavelittle ornosurfactantinthe alveoli, whichgives rise to averyhigh tendency forlung collapse(Griese,1999).
NRDS affects about 1percentof newborn infants;it isthe leading causeof deathin prematurelyborn babies(HallmanetaI.,2001).The respiratory disorderresultingfrom the damageanddysfunctionof surfactant during
injuriesorsepsis is called acuterespirator ydistre ss syndrome(ARDS) . ARDS has ahighrate of fatalit y,betw een 36%to52% (Seegeretal.,199 3).
Mutation sin the genesencodingsurfacta nt protein B andsurfactant protein C (SFTP 13and SFTPC, respecti vely)canleadto acute respir ator y failure and interst itiallungdisea ses.Respirat or yfailurein mature newb om s due tomutat ioninSP-B and SP-C encodinggenes is extreme ly rarebut has been obse rved repeatedl y as an inheritedcau se ofsevere respi ratory dysfuncti on (Nogeeetal.,2000).
Surfactantrepl acem enttherapyhashada proven role in the treatment ofNRDSand mayhave a rolein thetreatmentof patientswith ARDS.However, efforts tousereplacement surfactant totreatARDShave notbeensuccess ful thusfar;deacti vati onof thereplacementsurfactant red uces the efticie ncyo fe ndoge no ussurfactant therap yin thesepatients (Gunther eta l., 1999; Stevensand Sinkin,2007).
Surfacta nt baseddrugscan cont ainboththephospholipid and proteincontent. Syntheticsurfactants differremarkabl yfromnatu ral surfactantsin theirprotein compo siti on (Marraro,2004). The origin al commerciallyavailablesurfactant,(Exosurf;Glaxo Well com e),doesnot cont ain anysurfactant protein s.Clinical trialshave shown artificial surfactants aremuchmore effecti veif theyincludesurfactant protein s as compared toprotein-freepreparation s (Lewisand Veldhuizen , 2003).
Naturalsurfacta nts, deriv ed fromanima l lungsby organicextraction,
containsurfactant proteinsandaremuchmore effective inlowering the surface tension(Stevensand Sinkin,2007).
Thereare twotype of epithelialcovering the alveolarsurface; type IandtypeIIcells.TypeIcells formthe structureof analveo larwallwhile all lung surfactantcompone ntsaresynthesized by typeIIalveo larepithelial cells.Aftersecretion to thealveolarspace,lungsurfactant phospholipidscan form distinctphysicalstructuresfrommonolayerstomultilayers, ineluding tubular myelinand lamellar bodies,as well asvesicular,protein-lipid structures.Formationof thesestructuresismainly affectedby the stoichiometryof lipidsandsurfactantproteins,andphysical forces generatedduringrespiration (Whitsett,etaI.,2010).Althoug hthe compositionof lungsurfactantcan varyfrom onespeciesto another,lung surfacta ntiscomposedofaround80%phospholipids,5-10%neutral lipids
-mainly cholesterol- ,and8-10%proteins making up 5-6%of total
surfactant mass(Goerke, eta l.,1998).
The lipid fractionofmammalian surfactants(bymass)is composed of around80%phosphatidylcholine(PC),about half ofwhich isthe disaturatedformdipalmitoylphosphatidylcholine (DPPC)(Veldhuizen,et aI.,1998) .Different levels oftluidityduring the respirationcyelerequ ire a balance betweenthepresence ofsaturatedand unsaturatedlipids.Themost remarkabledifferenceofsurfactantcomposition inhumans in compariso nto other mamm alianmembr anesisthe unusualhigh contentof DPPCand
anionicphosp holipids,mostlyphosphatidylglycerol (PG) (Wustneck,et al., 2005).DPPCis ararephospholip id speciesin othertissuesbut evolut ionary hasbeen chose n.DPPC ' s saturatedchains packto ahighdensity at the air- waterinterfaceatphysiologicaltemperature andprovidea significant reductioninsurfacetension at the endof expiration(Hawcoaeta1.,1 981).
In mice and humans at37°C,the physiologicaltemperat ure,pure phospholipidshave a slow rate offilmformationand weak surfactant activity.Appropriaterate of film formation,stabi lity duringrespiration cycle andre-spreading capacities are dependent on thepresence ofthe surfactant proteins(WhitseUandWeaver,2002 ;Halliday,1996).
Surfactant proteinsdesignatedsurfactant proteinA (SP-A),SP-il, SP-C,andsurfactant proteinD(SP-D),play criticalroles invariousaspects of surfactantstructure,functio n,and metabolism (Perez-G il,2008).In general theyare dividedin two maingroups;thehydroph ilic surfacta nt proteinsSP-A and SP-D,and thehydrophobic surfacta nt proteins SP-Band SP-C.SP-Ais the most abundant protein componen tofs urfacta nt(Sueishi and Benson,1981) whichis associatedwithsurfacta nt phospholip ids (Schurchet al.,1992;Biet al.,2001).SP-Dis found inaverysmallamo unt inthelavageanddoes notassociatewith lipid containingstructu res (Haags manandDiemela,2001). The structuresof SP-AandSP-Dallow themtobindto diverseligandsunderlyingtheir role in the innateimmune systemat thelung surface(Crouchand Wright,2001).SP-B andSP-C
compose 1-1.5% of total surfactant mass.Thes e hydroph obicprotein s interactwith thelipidsin lung surfacta nt(Shi fferet al.,1993;Taneva and Keou gh,1 994 ).
SP-I3is theonly lungsurfactantprotein whose de ficiency is letha I (Clarket al.,1995; Tokiedaeta l., 1997). SP-I3ex ists in diffe rent classesof vertebra tes includin gfish (1ungfish),amphibians,reptilesand mammals; in humanthe encodi nggeneislocated onchromoso me2.Itbelon gstothe familyof saposin-like proteins,which possess a specia l fold of around 80 amino acidsconta ining amphipathi c alpha-he licesand three conserve d disu1phidebridgesinconserve d position s (MunfordetaI., 1995). All sapos in-like proteinshaveactiviti esrelat edto interaction with phosp ho lipid memb ranes (Patthy,1991).
Thematur eSP-I3 isolated fromlunglavagesis ahighl y hyd roph obic peptidewith79 aminoacids,with-8 kDa molecularmass;the functiona lform is ahom od imer.Itpossessesthree covalent intramole cular disulph idebon dsperm onom er and an additi onaldi sulphid ebond stabili zin g thedimer (Serrano etal.,2006)(Figure 2).ltis synth esized as aprecursor (38 1ami noacids)followe d byproteolytic cleavageofN -andCste rmi nal prop ept ides in several different stepsthatresultinthe matur e seq uence (Brasc hetal.,2003 ;Uenoeta l.,2004).
~ . : \ .
,:
N N
~ .. : \ ' :
,FPIPLPfWLIRALIKRIOAMIPKG~LAYAYAOyil!!l!VPLVAG§IIoILAERYSY!LL[JTLL§RMLPOl\iIRLVLRIsMI
Figure2. Schematic diagram of mature SP-BandSP-B homodimer.
Disulfidebonds connecting the predictedhelicesareshown byd ashed lines.
The fourthdisulfide bond (red dashedline) stabilizesthedimer.Theamino acidsequence andexpected helical regions(boxes)areshown, cysteine residues arehighlightedingrey.
The synthesized precursor SP-B istransported from the endoplasmicreticulum tothe Goigiapparatusand then tomultivesicular bodies whereit ispackedinto lamellarbodies.Proteolyticprocessing ofSP- B occurs in typeII pneumo cytesduringtheexocytic pathwayof surfactant.
The activeSP- B peptideisstored with SP-Cand surfactant phospholipids in lamell arbod ies(Voorhout et al.,1992;Korimilli eta 1.,2000).
Several mechan ismshavebeen suggested to underlie SP-B's function includingenhancingtheinterfacia ladsorptionofphospholipids fr om the hypo phase tothe air-water interface (Cruzetal.,2000;Schram and Hall,2004),stabili zing compressedfilms at the alveolarsurface(Krolet al.,2000),facilitating there-spread of thesurfactant mater ialduring expansionand providin glowestsurface tension during respiration cycle.It hasbeen proposedthat SP-Bfunctionsas a linkfro m bilayers (hypophases) to the monolayer (air-wa ter interphase) and back (Tanevaand Keough, 1994; Zaltashe tal.,2000)(Figure3).
... ...
... ...
Figure3.A modelreprese nting the roleofSP-Bduringcompression(a.
pointing in arrows) andexpansio n(a.pointing outarrows)ofbreathing cycle.S P-B mediatesbilayer-bilayera nd bilayer-monolayertransitions.
Previo usworkshave show n that SP-B fragm ent s can retain simi lar activitiesto thefull-leng thSP- B.TheN-term inal hal f of SP-B consistsof aminoacids 1-37,and has ahighnumber ofhyd roph obi c andcationic resid ues thatprovides lipid-interacti onproperti es.C-terminal basedpeptid es alsoshowedin vitro andinvivosurfactantactivities(Kang etal.,1996;
Baatzetal.,199 1;Ryan ct al.,2005). Enhanceme ntof oxygenationand lung compliance, dynam ic re-spreadin g in anima l model s, membr ane permeab ilizati on and liposom elysishavebeen repo rted for N- or C-termi nal helical fragm ents of SP-B (Revak et al.,1991;Walth er etal.,2002)(Figure 4).
N _ _ . _ C SP·BNTt RN
C~Minl'B N~
Figure4. SchematicdiagramofSP-B peptidessynthesized based on the prcdictedhel ical segm ent s of SP- B. SP-BNTERMand SP-BCTERMaredesign ed basedon the N- andC-term inal helicesof SP-B respect ively.Super-Min i-B
and Mini-Bare N-andC-term inalcontaining peptid es.The disulfid ebond s connecting thehelices areshown bydashedlines.
Arecentin vivostudy(Waltheretal.,20 10) assessedbiological activitiesof a groupofSP-B pcptides;Mini-B. Super-Mini-BandN- terminal fragmentof SI'-B(SP-B,_s).Mini-Bis a 34-residue peptid ethat consistsofthe N-and C-terminal predictedhelicalregionsofS P-B.Super- Mini-B is a41-residuepeptid e with two internal disulfid ebondsthat contains theN-terminaI7-residueinsertionsequenceand thepredicted N- andC-termi nalhelices of SP-B.Theseregions are connectedviaaloop as wellastwo disulfidebonds(Waringetal.,2005).Super-Mini-Bcan be consideredas amodifiedMini-B since the only difference istheprese nceof theinsertion sequence(SP-B,_7)(TableI).
Table!.Aminoacidsequencesof SP-B,Mini-B andSuper-M ini-B.
Disulfidebond s areformedbetween cysteine residueshighlighted by gray
Protein AminoaddstqUel'lCt
IP·8 f~PlPY(WllAAUKRKlAMIPKGA----lAVAVAQVtRWPlVAGGICQClAERYlVlUDlUGRM lPQ.l1RlVUts-M
Miri-B
-
.- - ii\lRAiiKRKlAMIPKG - -- - - -- -GRMl.PQ.\1RLvu lS-MS~-Mi"' B m~tMUKRKlAMIPKG---GIlMl.fQLI1RLVUcs_M
Inthis study(Walther,etal.2010),lunglavaged ratsweretreated by artificia lsurfactants composedoflipids aloneand lipidsplusSP-B, Mini-B,Super-Mini-B andN-terminalfragmentof SP-B(SP-BI.s).The study included the measurementof arterialoxygenationand lungdynamic complianceinlunglavagedratswithARDSandcontrols.The treatment with lipids alonedidnot improve theoxygencontentin blood.Lipids+SP- BI•8improveditonlyslightlymorethanlipids alone. Lipids+Mini-S producedhigher oxygenation levelsthanlipids+SP-B.Lipids+Super- Mini-Breachedthehighest valuefor arterialoxygenation(Figure5).The results suggestthattheN-terminalinsertionsequence(SP-BI•7),in combinationwith theN-andC-terminal helicesof SP-B,provides greater surfactantactivity than either the N-andC-terminal helices alone,orfull length SP-B .
Lungdynamiccompliance isrelatedtothevolumeoftheairthat is exchanged duringrespiration.Measurementof this value byWaltherand co-workersindicatedasimilar relative activity ofthe peptid escomparedto the oxygenation measurements.Theonly differencewasthatlipids+Mini- B hadalower activity thanlipids+SP-B(Figure5).Thisresultindicates thatthe presence ofN-terminal insertionsequence(SP-B1_,)maybe important forlung expansion-compression.Italsosupportsthepossibilityof adiffer entmechanismof function of SP-B for oxygenation compared to lungdynamics.
Oxygena tio n OynamicCompliance
1t;:
,oo ;;~ 06
¥
soc ~ 5S8" 00 ~ 5
~ J ~ 05
~ 10
j
0-c 100
i
0 "Figure5.Arterialoxygenationanddynamiccomplianceinrats subjectedto removaloflung surfactant by in vivo lavage.Exogenouslysurfactant replacement was administered by syntheticsurfactants preparedfromlipids onlyand lipidsplusnative SP-Bor SP-B peptid es.Syntheticlipids+1.5 mol%Super-Mini-B ,Mini-B,or SP-BI_8•syntheticlipids+1.5%porcine SP-Bandsynthetic lipidsaloneasa control.Arterial partialpressure of oxygen(Pa02)and dynamiclung compliance(mUkg/ cm H20;calculated bydividingtidal volumelkg body weightbychangesinairway pressure) are shownasa functionof time aftersurfactantadministration.The plots are self-prepared usingthe publisheddata of Ref.Waltheretal. 2010.
Theknowledgeofastructure ofa proteinprovides informationon its molecularmechanismof function.Theexact three-d imensional structure of SP-B is stillunknown.Structureof someSP-B peptid eshavebeen determ ined bysolutionnuclear magnetic resonance(NMR) experime nts;the N-terminalpeptide(SP-BII.25)(KurutzandLee,2002),and the C-tenn inal peptide(SP-B6J •78)(Booth,etaI.,2004) (Figure6).The highresolution structureof Mini-B has also been determined(SarkeretaI.,2007)(Figure 6). Inorder todefin ethe importanceof the N-tennina l7 residuesofSP-B (SP-BI_7),results of structuralstudieson Mini-B and Super-Mi ni-B.
particularly NMR experiments,werecompared.
Figure6.Thehigh-reso lutionst ructuresofth ree terminalfragmentsofSP-B determ inedbysolutionNMR. The fragmentsare SP-BNTERM(SP-BI I-25)in
methanol (PDB lD I KMR) (KurutzandLee, 2002),SP-BCTERM(SP-B6J.7S) insodium dodecyl sulfate(SDS) micelles (PDB ID 1RGJ ) (Boothetal., 2004),reducedMini-B(SP-BS-25+6J.78)in hexafluoroisopropanol (HFIP) (PDB ID2JOU)andoxidized Mini-BinSDS micelles (PDB ID2DWF) (Sarkeretal.,2007).The disulfid ebondsare not shown.
NMR iscommonl y employed instudying threedimensional structuresofproteins. Oneof theadvantagesofNMRspectrosco py isthat datacanbe acquired insolution.Studying the structureof proteinsin solutionwith thepossibilit y ofadj ustingconditions(suchastemperature, pHandsaltconcentration)allow usto closely mimiephysiological conditionsandstudyin vivo functionsof proteins(WUthrich,1986).
NMR is apropertythatmakesparti cularnuclei inamagneticfield absorbenergy fromtheapplied electromagnetic pulse and radiatethis energybackout.These nuclei arecalledNMRactive nucleiory,spin nuclei.Theenergyradiatedback outfromeachnucleushasa specific resonancefrequencydependin g onthe strengthof themagnetic fieldand the environmentsurrounding thenucleus (Poehapskyand Pochapsky,2007). In proteins purified from natural sources thereis only oneV,spinnucleus,IH.
TheaxesinNMRspeetraarefrequen cy,normallyrepresented as chem ical shift.Thechemicalshift describesthedependency of the radiated
energy level of amagneticnucleus on the electronicandchemica l environmentsurrounding thatparticularnucleus. Solution-stateNMR experime ntsprovideinforrnationabout the structureofa protein based on twotypes of correlationsbetweennuclei;correlations betweennuclei with -3 covalent bonds of eachother(from total correlationspectrosco py (TOCSY)experime nts),andinteractions betweennucleithat are closeto eachother throughthe space conformatio n,but not through the chemica l bond (from nuclear overhausereffectspectrosco py(NOESY)experiments) (CavanaghetaI.,1996).
Oneofthelimitations of solutionNMR isthe size issue.Lar ger moleculeshave slowertumblingratesandshorterNMRsignal relaxation times.This reducesthe sensitivityof received signalsand resulttoweak peaks. Also more complexitywillintroduce to a spectrumbylarge size molecules because there are more NMR-active nuclei and moreinteractions among them (Yu,1999}.Itishardto getinformationofthe structurefrom broad and low intensity peaks.Thelimitation of solution-stateNMR in studying hydroph obicproteins suchas SP-B isthat, thisclass ofproteins cannotbebroughtinto a sutlic ientlyconcentratedsolution duetotheir highlyhydrophobicnature (LawsetaI.,2002).Detergent or lipid micelles canbeusedto mimicthe lipid environment tosolubilize theseproteins.A micelleis anaggregateofa mphiphilic molecules,inapolar solvent( mos tly water}. These amphiphilicmolecules containahydroph ilichead groupand
onelong ortwo shortacyl chains.Whentheconcentration ofthese molecule sin wateris greater than acertain value,known asthecritical micellar concentration(CMC), sphericalstructurescalled micelles are formed which thehydrophilicheadgroups areexposedto watermolec ules andburyingthe acyl chainsinside.Neverthelesssolubilisation of hydroph obicproteinsby lipidcontainingsolutions isnot helpfu I enoughfor NMRstudiessince theproteinsin complexwithlipidstumble veryslowly and don't give riseto detectablepeaksinsolutionNMRspectra (Sanders andSonn ichsen, 200 6).
Solid-state NMR worksbased on the resonance ofNMR-active nucleiin themagneticfield.In thistechnique,interactionsbetween nuclei areorientation dependentand takeplacein mediawith no or little mobility.
Itisan alternative technique togive information about theproteinstructure.
Howeverit doesnotprovidehigh resolut ion structural datain com parisonto solution-state. Solid- state NMR is also a valuabletooltostudy local dynamics,kinetics and thermodynamicsof protein-lipidinteractions in protein-membr ane systems (Laws etaI., 2002).SinceSP-B isa membrane associated protein, studying SP-B peptides in lipid bilayers by solid-state NMR providespreciousinformationabout SP-B/lipidinteracti ons.
Inspite of SP-B's importance,attempts at recombinantexpression ofthe full-lengthprotein and chemical synthesis ofanear-fullpro teinhave
not succeededyet,mainly dueto its highdegree ofhydrophobicityand the prescnceo fthree intrachain disulfidebonds. However,fragmentsofS P-B retain muchof thefunctionofthefulllengthprotein (Kangetal.,1996;
Baatzet al.,1 991;Ryan et al.,2005),making themhelpful tounderstand the mechanismofthe full lengthprotein. Additionally, peptides are of interes t astheypose substantialadvantagesoverfull length proteins to useas therapeutics,e.g.for NRDSor ARDS.
The main objec tivesofthis workare toproduce full/nearfull length SP-Bsuitablefor NMRstudiesand to understand the roleofthe N-terminal 7residuesofSP-B.SP-B based peptidesencompasssimilaractivi tiestothe full length protein,this factraisethehypothesisthatparticularstructural featuresof SP-Bare responsibleforparticular activitiesof SP-B.In this research,structuralstudieson SP-B peptides;Super-Mini-Band N-termin al insertionsequence ofSP-B(SP-BI _7).The research was performe d using solutionandsolid-state NMR spectrosco pyin order to characterize the structuralchangesand membr ane interactio n properties induced bythe by presence ofN-terminalinsertionsequence inthecontextofSP-B peptides.
Moreover,recombinantexpressionofSP-Binanattempt toproducethe full-lengthproteinandoptimizingexpressionconditio nswere investigated . In thiswork also production and expressionofa nove l near full-IengthSP-B peptide; N-terminaldeletedSP-B(Nd-SP-B)wereexamined.Theoverall
resultofthisresearchprovides a stro ngfoundationforfuturestudieson the production,conformationand interactions offull-lengthSP-B.
2. 1.Sequ en ceidentifi cationof SP-Bencoded plasmid
Theplasmid containingfull-lengthSP-B gene wasdesigned and synthesized bymembers oftheJesusPerez-GillabatUniversidad Complutense(Spain).Startingwiththisplasmid, it wastransformed into DI15aE.coli cells.The plasmidwas amplified in thesecells,thecellswere harvested andtheDNA isolatedusingthe extractionand purification procedures ofQIAprep®Miniprepkit(QIAGEN Inc. Mississauga,ON).
Bacterialcellswereharvestedfrom a 2mlcell culturebymicro centrifugation.The pellet wassubjected to thealkaline-SDSlysis procedure.
The solutionaddedto amicro column filledwithsilicaandequilibrated withahigh saltconcentration. DNA adsorbedonto thecolumnand contaminantswere removedby a simplewashstep.The bound DNAwas elutedin water orTris-EDTAbuffer.Thepurified DNAwas sequenced by the Genomicsand Proetomics(GaP) Facility,(Memo rialUniversity).
2.2.Transf ormati onof tile plasmid totile exp ressioncell line Afterconfirmationof sequences,Iftl(0.1 ng/ul) ofthepurified DNAwas added to a 50ftlaliquotofcompetentC43(DE3)cells. The
transformationreaction wasincubated on icefor30 minutes.Theheat shock wasappliedfor45 secondsat 42°C and thenthe reactionwasplaced on ice for 2minutes. 500fllof lysogenybroth (LB) mediumwas added tothe transform ationreacti onand incubatedat37°Cfor 1 hour.The LB medium consisted01'10g/Ltryptone,5g/L yeas textract, 10g/L NaCl, pH 7.After incubation,the transformati onreactionwasplatedonLB-ampicillin(100 flg/m1)agar plates and the plates were incubatedat3 7°C for 16 hours.Later in myworkto optimize thecell growth, I haveusedterrific broth(TB) mediawhich is aricher thanLB.TB mediumisconsistedof 12giltryptone, 24gilyeastextract,9.4gilpotassiumphosphate (dibasic),2 gil potassium phosphate(monobasic).ThefinalpHwas adj ustedat7.
2.3.Recombinant expression of'full length surfactant protein B
An isolated colonyon thetransform edplate was selected and placed in10 ml LB-ampicillin(100 ug/ml),The mixturewasincubated overnight at37°C.The overnightculturewas added to750mlLB-a mpicillin broth.
Cellsweregrownat30°Cuntilthe001000 reached 0.7,and then isoprop ylp- O-I-thiogalactopyranoside(IPTG) (I mM)was added to induce expression ofS I'-B.Alier 4 hours incubation.the cells wereharvestedby cent rifugation at 4000gfor 10 min. Thepelletwasresuspendedin a minimized amountof resuspen sionbuffer(10 mMTris HCIpH7.9, I mMethylenediaminetetra acetic acid (EOTA), 0.01M phcnylmcthanesulfonyltluoridc or
phcnylmethylsulfonylfluoride(PMSF))and kept in -20°Cfreezeruntil
Inorder tobreakthe cell memb ranes, pelletsweresubjected to French press atroom temperature,sonicationwhilethesamplewaskept on ice.TheFrenchpresscontainerandthesonication probewere cooled down byice beforeusage. After thecellrupture,Irisbufferedsaline (TBS)1%
lauroylsarcosinedetergent (TBS=50 mM Tris,150 mMNaCI,pH 7.6)
centrifugedat 18,000 g forlOmin.The fusionprotein waspurified fromthe supernatantvia immobilized metal affi nitychromatograp hy.The cobalt containingresinwasusedto make a His-Trapcolumn(TALONMetal AffinityResin,ClontechLaboratories,lnc.MountainView, CA,USA).The columnwasequilibratedwithTBS1%lauroyl sarcos ine bulle r. The supernata ntwasloaded ontothe cobaltcolumnandthecolumnwas washed withTBS, 1%lauroyl sarcosine,75 mMimidazole. Theproteinwas eluted fromthe columnbytheelutionbuffercontainingTBS, 1%lauroylsarcosine and 500 mM imidazole.Theeluted protein was dialyzedagainstwater to remove imidazo le,bufferand detergent.After dialysis,the solutionwas
2.4.Production&recomb inant exp ression of tile Nsterm in al deletedSP-B
Site-direc ted mutagenesis was appliedon thefull-length SP-B containing plasmid.Stratagene'sQuikCha nge®Site-Directed Mutagenesis kit(AgilentTechno logiesCanadaInc. Mississauga, ON) wasusedtodelete theN-termina lfragmentof SP-B.Theforwardandreverse primers were designedpersonally based onthefullsequenceofthe plasmidand consideringthe desireddeletion mutatio n.Bothofthemutagenicprimers containedthe mutation andanneal tothe samesequenceonopposite strands oftheplasmid (Table2).Theencodingsequenceof sevenN-terminal residuesof SP-B (FPIPLPY)was absent inmutage nicprimers.The synthes izedmutagenicprimerswereorderedand purchased from Sigma- AldrichCo. (St.Louis MO).5f1lof
to-
reaction bufferwasmixed with2f1l (5-50ng) dsDNAtempl ate (the purifiedplasmid DNA ofthebacteria before themutation), 2f1l(125 ng) forward primer,2ul (125ng)reverse primerandIf1ldeoxyribonucleoti detriphosphate(dNTP) mix.Double distilled water was addedtobring thevolume to 50 f11. ThenI f1lof Pfu DNA polymerase(2.5U/ul)was addedto reaction mixture.Polymerase chain reaction (PCR) wastousedto amp lifythe mutationreaction. The PCR reactionwas asfollows:a pre-denaturation step,30 seconds heat treatmentat95°C.Thereaction was followed by18 cyclesof denaturatio n,30 secondsat95°C,annealingfor I minute at55°C andextensio nfor 20minutes at68°C.Thesecond part ofthePCR reaction was repeated for18 cycles.After completionof thereaction,I f11of the Dpn
I restricti onenzyme(10Ulfll) was added to the amplifi cationproduc tand incubated at 37°Cfor 1hour to digestthe parental(unmutated)DNA.DpnI isanendonuclease,spec ifi c for methylated and hemimethylatedDNA.Itis usedtodigesttheparentalDNA.DNAisolatedfrom almostallE.coli strains ismethylatedand therefore susceptible to Dpn I digestion.The sequencingofmutation productsconfirm edtheefficiency of Dpnl.The mutated product wastransformedintoC43(DE3)E. coli cells.The same procedure s,asdescribedin2.1to 2.3,wereapplied to prepare asample for DNA sequencingandexpression.
Table 2.Thesequencesof forward and reverseprimersdesignedforsitedirected deletionmutation on theN-terminalofSP-B.
Primers Thesequence
Forward 5'-GTTCC ACGG GGCCC A TGCTGG CT CTG CA GG Primer
Reverse 5'-CCTGC AG AGCC A GCA TGGGCCCCGTGGAAC Primer
2.5.Ident ifi cation ofthe expressedpeptide
Therecombinantlyexpre ssedpeptideswereidentifiedusing westernblottingbyimmunoblottingof anti-histidineantibody. The histidine
tag wasinsertedat the N-terminal of the desired peptide,thepurposewas to facilitatethe purificationprocessthroughmetal affinitychromatographyand also theanti-histidineantibody wasusedto detect theexpressionof the desiredpeptide.Since the desiredpeptide is a novel constructusinga synthesizedantibody isnot convenienl. Forwestemblotting,firstthe eluted protein from columnchromat ogram(described in 2.3) was loaded on a SDS-I'AGE,sodium dodecylsulfatepolyacrylamid e gel electrophoresis, gel. TheSDS-I' AGE gel consists of bothstackinggel andseparating gel.
Gels were solidified ina gel caster.To preparea 12%SDS-I'AGE gel, first the 10miseparatinggel waspoured and polymer ized.The separating gelis made of 4 ml30% acrylamid emix (acrylamide 29.2%+0.8% N,N'- methylenebi sacrylamid e), 6ml 0.75M TrispH 8.8,0.2 %SDS,100 fllIO%
ammonium persulfate (AI'S)and20 fll tetramethylethylcnediamine (TEMED).The5mlstackinggel wasmade of650fl130%acrylamidemix, 4.35ml 0.13M Tris pH6.8,0.12%SDS,100 ul 10%AI'S and 10 ul TEMED.Chemicals werepurchasedfrom Sigma-Aldrich Co.,St.LouisMO andFisher ScientificCo. Ottawa,ON.After the separating gel was thoroughly solidified,the stackin g gel solutionwas addedtothecaster and a plasticcombwasinsertedtoform wells.Then thegel wasplacedina runningchamberfilledwith IxSDS running buffer (25 mM Tris pH 8.3, 250 mMglycine,0.1%SDS).The proteinsamplewasdissolvedinsample buffer(65mM Tris-H ClpH6.8, 1.3%SDS,13%glycerol,1%p-
mercaptoethanol,0.02%sodiumazideand 0.02%bromophenolblue), boiledforIminuteand loadedinto oneofthe wells.The electro phoresisat 180 V wascompleted whenthe samplebufferdye (bromopheno l blue) reached tothebottom ofthe gel.
Electrophoretic transferof theproteinfromthe geltoa polyvinylidc nefluoride(PVDF) memb rane (Mi lliporeCorporation,MA, USA)wascarriedout by electrophores isat60 Vfor120minutes.The mem brane was thenkeptfor I hour at roomtemperature in a solutionof 5%
non-fat milk in TTBS;TBS (20mMTris, 0.5MNaCI, pH7.6)and 0.05%
Tween-20.Themembranewasthen incubatedwitha primary antibody against thehistidi netag,monoclonalMouseIgGI,at roomtemperature for 2hours,and thenthe antibodysolutionwas washed from membran ein TIBS for several times.The membrane was incubated with the seconda ry antibody (po lyclonal GoatAnti-Mouse IgGAlkaline Phosphatase conj ugated)for 1 hour at roomtemperature followed by severa l wash steps.
The dilutiontime fortheprimary antibody (anti-h istidine)wasl:5 000 ( 1fd in5000 ul) and the secondaryantibody is1:3000.Antibodieswere purchased from Sigma-A ldrichCo.,St.Louis,MO.To visualize the bound antibodies,BCIPINBT color reaction was employed. BCIP (5-bromo-4- chloro-3-indolyl-phosphate) in conj unction with NBT (nitro blue tetrazolium ) areusedas chromoge nicsubstratesforthe colorimetric detection ofalkalinephosphataseactivity. BCIP as acolor generatorand
NBTforsignalenhancement,they areidea l for immunoblottingwhile alkaline phosphatase is conj ugated tothe secondaryantibody.5mlof alkalinephosphatase buffer (l OOmMTris-HCl [pH9.0], 150mM NaC!, ImM MgCI2), 75mg/mlNBTin70%dimethylformalmamide (DMF)and 50 mg/mlBCIP in 100%DMFwere mixed,the solution wereappliedtothe PVDF membrane that wastreated with the primary antibody in theprevious step.NBTandBCIPwerebought from Sigma-AldrichCo.
2.6. Chemicalsy nthesis and purification ofNstermlnal fragment of SP-B
N-terminalinse rtionsequence of SP-B(FPIPLPY) wasproduced bysolid-phasesynthesisusing 9-fluorenylmethyloxyca rbonyl(Fmoc) chemistry.Aminoacidswereweighed outin5xexcess andplaced into aCS Bio peptidesynthesizer(modelCS336X, CSBio Company Inc,MenloPark, CAlusing0.43gofa0.47mmol/g Rinkamide resin (CSBio Company Inc, Menlo Park, CAl .Dissolut ion andde-blockingof aminoacidswere facilitated using a 0.4 M I-hydroxy-benzotriazole dissolved in dimethylformamid e (DMF),anda 20%peperidine/DMFsolution(Sigma- AldrichCo.,St. Louis,MO).Theresin waswashed with DMF.After completionofsynthesis,theresin containing synthesized SP-BI_7was transferred to a10 mlsyringeequippedwithafilter,andwashedwith methanol.Thentheresin was air driedfor 30 minutesfollowed by vacuum
dryingtor 60minutes. The peptide was cleavedfrom theresinby a cocktail consistingof9 ml trifluoroaceticacid( TFA), O.25ml l,2 -Ethanedithiol, O.1 ml thioanisole (Sigma-AldrichCo.),and 0.25ml distilledwater.The peptide wasprecipitated fromthesolution by additionof50mlof-20°Cdiethyl ether. Theprecipitatewaspelleteddown by centrifugationat 4°C,4,000 rpm,5 min. The precipitantwas collectedand allowedto air dry overnight.
Purificationprocedure wascarriedout byusinghigh-pr essure liquid chromatogra phy(HPLC)(Varian ProStarHPLC,VarianInc.,St.
Laurent,QC).ThelIPLCwas equippedwithareverse-phaseDYNAMAX C8 preparatory column (Varian lnc., St. Laurent,Qc) , anda spectro photomete roperatingata wavelengthof 215 nm.The peptid e was eluted using anacetonitrilegradient(80/20% HPLC gradewater/acetonitrile -0/100% acetonitr ile;Sigma-AldrichCo). The mass andsequenceofthe peptidewere confirmed throughmatrix assisted laserdesorpti onioniza tion- time offlight mass spectrometry(MALDI-TOF MS)by Genomicsand Proteom ics(GaP) facility(MemorialUniversity).
2.7.Solution nuclear magneticresonance(NMR)
The structureofSuper-Mini-B andSP-BI_7were assessed by solution-statenuclea r magneticresonance (NMR). Thepeptideswere investiga tedinanorganicsolvent(HFIP)andamembr anemimick ing environment(detergent containing solution). I mMSuper-Mini-Bor2mM
SP-BI_7weredissolved in 40%HFIP(hexa fluoroiso propano l),50% H20 and 10%020with 0.4mM 4,4-dimethyl-4-silape ntane- l -sulfonicacid(OSS).
Fordetergentcontainingsolution, I mMSuper-Mini-Bwere dissolvedin 90%H20,10%020,0.2mM DSS, and ISOmMdcuteratedSOS(Sodium dodecyl sulfate).Valueswerethe same for 2mM SP-BI _7SOSsample except thepeptide wasdissolved in300mM deuterated SOS. Oeuterated SOS,OSSand 020werepurchased fromCambridge Isotope Laboratories, Inc.(Andover,MA).Forallexperiments, OSS wasusedto obtain theproton referencesignalat 0ppm andwater-gatewatersuppressio nwasused witha 3-9-19pulse.Alltwodimensional experimentsused arecycledelay ofl second.One dimensional (10)and two-d imensional (20)NOESY,TOCSY anddiffusio norderedspectrosco py(OOSY)experimentswere carried out usinga TXI probe onaBruker Avance600 MHz atCentreforChemical Analysis,ResearchandTraining(C-CART) (Memoria lUniversity).
For I DIHNMR,32 scanswererecorded.Amixingtime of 200ms was usedwith 160 scans forthe20NOESY.For the 20TOCSY,aD1PSI2 sequence wasused,andthe mixing time was 80ms,with 128 scans. Acquisitionofspectrawasperformedusing TopSpi n(Bruker,Milton, ON).
Spectrawereprocessed using iNMR(http:// .inmr.ne t)and analyzed using SPARKY(Godda rdand Kneller,2005).ForNMRstructural studies, eachpeak needsto be assignedasa specificnucleusin the molecule under investigation.Resonanceassignmentsalso have tobe sequence-
specific,i.e.,each peakmustbe assigned to anucleus orcorrelationof nuclei in a particularaminoacidin theproteinsequence.IHpeaks were assigned to a chemicalgroup type on thebasis of their chemicalshifts.
Amide protons(HN) resonatebetween10.0 and7.0ppm, thebackbone a- protons (HaorHA)resonatebetween 6.0and3.5 ppm,the aliphaticside- chainprotonsresonatebetween3.5 and1.0ppmand themethylprotons resonateatchemica lshifts less than 1.5ppm (Cavanagheta l., 1996).
The diffusionexperimentswere performed on 150mMSOS in H20 and 020 withImM Super-Mini-BatpH 5 and30°C, with the same sample used for othersolutionNMRexperiments.Thepulsesequencefordiffusion measurement used stimulated echo with bipolar gradient pulses,followedby 3-9- 19pulsefor watersuppressio n.OOSY spectrawerecollected in32 steps,attenuatingsignals to about5% of theinitialvalue byincreasingthe gradientstrengthfrom2%to 95% ofthe maximumamplitude, for a constant diffusiontime of100 msandanoptimizedgradient pulselength01'4ms.
Thespectra wereprocessedusing iNMR and theditTusion constants calculated using the"DOS Y" packagein DOSYTooIBox08 (Nilsson,2009).
16-doxyl-stearic acid (16-DSA)was addedfrom a 2mMOSA in NMRSOScontaining butlerto Super-Mini-BandSP-B1.7samples,thefinal concentrationof DSA was 0.6mM.The samples wereusedfor OSA- TOCSYexperiments.DSA waspurchased fromSigma-AldrichCo., (St.
LouisMO).
2.8.Solid-state nuclear magnetic resonance (NMR)
The oriented lipid/lipid-protein sampl eswerepreparedusingRainey andSykesprocedure(RaineyandSykes.2005). Muscov itemica(gradeV-a.
dimension: 75x25x0.26mrrr'),purchased from Structure Probe (West Chester, PAl, was cut into small piecestoprepare12plateswith dimensionsof12.5mmx5 mmand thickness of -40 urn.Deurterated1- palmitoyl-2-0Ieoyl-sn-glycero-3-ph osphocholinc, POPC-dll, and 1- palm itoyl-2-0Ieoyl-sn-glycero -3-phospho-(I'-rac-glycerol), POPG, were purchased fromAvanti PolarLipid(Alabaster,AL).For thelipid only sample,4 mg ofPOPC-dl!and POPG ata ratioof7:3were dissolvedina solventcomposedofC HJOH/CHCll(1:1byvolum e).Forthe lipid-peptide sample,I mol%(byweight) ofSuper-Mini-Bwasadded tothelipid- organicsolventsolution. The solution,with atotalvolume of-250~I,was spread over micaplates.Theplates weredriedfor-2h in a fumehood and thenplaced in a vacuumchamberovernight.Afew microliter s of deuterium depleted water werespread oneachplateand the plates were keptina hydration chamberwithsaturatedammonia phosphatesolutionat 4°C for 3 days.Thentheplates werestacked together,wrappedwith aplasticfilm, andsealed withpolystyreneplastic.Sampl eswerestoredat 4°C.The2H spectra were acquiredatatemperature of296Kona spectrometer operated ata resonancefrequencyof6 1A MHz.
2.9.Circulardichroism(CD)spectroscopy
Samplesprepared forstructuralstudies bysolutionNMR (preparationdescribed in2.7) were alsoinvestigatedby circulardichroism (CD) spectrosco py.The secondarystructural characteristicsofSuper-Mini-
£3,Mini-Band SP-BI_7were assessed inaqueous(HFIP containin g samples) and lipid mimicking environment(SOSsamples).TheCOspectra were
obtained using aJascoJ-810spectropolarimeter.Experiments were carried outat25°Cusing1mm quartz cuvette.Thespectrawere acquired asthe resu!tof 4 repeatedscansfrom260to 200 nm,using scan intervalsoflnm andanintegration time of 1s at eachwavelength,Secondarystructure content wascalculatedfrom the spectrausing COProsoftware (http://lamar.colstate.edu/-ssreeram/COPro) developedbyWoody andco- workers (Sreeramaand Woody,1993).CD valueswere converted to mean residueellipticity (MRE). Basis set 2 of the COProsoftware wasused and analysis wascompletedusing CONTIN/LL method (Johnson,1999:
Sreerama andWoody, 2000;2004 ).
3.1. Detection of expressed recombinan tfull length SP-B
The starting point of this study wasthe plasmidDNAprovided by JesusPerez-Oil' sresearch groupat Universidad Complutense (Spain) .The plasmidwasdesignedto contain thefull-lengthSP-Bgene fusedto
staphylococc usnuclease(SN). SN as afusionprotein is attached to SP-B through alinker sequence .Arecogniti on site for proteasedigestion (thrombin digestion)wasprovidedto separate SP-B from thefusionpart.
Sixhistidineresidues wereadded tothe N-ter minusofSN sequence to facilitatepurification of the expressed proteinbymetal affinity chromatography.
Theplasmid was amplifiedin D115uE. colicells inanovernight cnltureofLB media.TheDNA sequencingof multiple clonesconfirmed the expectedsequence forthe designed plasmid [DNA sequencing-g enomics andproteom ics (GaP)facility,MemorialUniversity].The plasmidwasthen transformed into the C43( DE3) cellline.This specificstrainofE. coli is genomically modifiedtoprovidethe optimumoverexpress ionof toxicand hydrophobic proteins( Dumon-Se ignoverte ta l.2004).
Transformed cellswerecultivated inLB mediaand inducedby1 mM [PTG. After the overnightinduction,cellswere centrifugeddown and the pellet was collected.10% Laurylsarcosinewas added tothepellet beforethe celldisruption procedure inorder to enhancecell membr ane disruption and to solubilise the hydrophobicexpressed protein.Cells were thcn disruptcd by French press andsonication.
The expressedproteinwaspurified from the cellextract bythe affinityofthehistid inetagto acobalt column.Detection of the purified protein onaComassie stainedgelwasnot promising becausethree bands
were detected in the purifiedsample while wewere expecting only one band (Supplementary material, Figure St.).InWesternblotanalysis a single immunopositivebandwas observed(Figure 7),asprobed byan antibody specific for the histidinetag (sinceanantibodyagainst SP-B isnot available).Theapparent molecularmass ofthe expressedfusionproteinwas estimated bycompari sontothemolecularmassmarkersrunon the same SDS-PA GE gel usedforWesternblottin g.The expect edmolecularmassfor SN-SP-B is-27 kDa whiletheestimatedsize ofthe detectedbandis approximately 22 kDa.
Figure7.Westernblot analysisofthe cellextract harvestedfromthe overnight culture of cellsencod ingSN-SP-Bplasmid. a)The band
represents antigen-antibody reactionupon applyinganti-Histidine antibody.
The histid inetag islocated at the N-terminalof SN-SP-Bsequence.
While alarge diffe rencein apparent molecular weightvia SDS- PAGEisnotunexpected for ahydrophobic protein(seeDiscussion )- characterizationofthe expressed construct by analternative method was desirable.For this,MALDI-TOF MS wasused[genom ics and proteom ics (GAP)facility,Memorial University].The purified sample wasdigestedby trypsin thatcleavespeptid e chainsat the aminoacids lysine orarginine, except when eitheris followed byproline.The trypsindigestionis ahelp ful step to allowanefficient tandemmass spectroscopy.Tandem(MS/MS) mass spectrosco py is a techniq uetousemorethan one analyzerandso can beused forthepeptidesequencing.First themass spectrometerionizatio n source(MAL O!)ionizesthesamplemolecules.The mass analyzer (TOI'analyzer)separates the ions formedin theionization sourceofthe mass spectrometer based on theirmass-to-charge (mlz)ratios.In the tandem-t ype mass spectrometer,twomass spectrome tersareconnecte d in tandem . Between thetwomass spectrometers is an ion selectorforselecting onlya specificion.Forexample inTOFTOFmass spectrometers,ionsare separatedin the first TOFstage.Justbefore a specific ionreachesthe ion selector, the selectorvoltage isturned off.Then,after theion of interest passes the ion selector,its voltage isturned onto allowonly theion of
interest topass. TheMS/MS spectrumcan provide informationaboutthe aminoacidsequenceofthepeptide ofinterest.
Resultsconfi rmed the sequenceof somefragments fromthe fusion protein andSP-B(Figure8 andTable3).Whilethepresence ofthe N- termin al residues ofSP-Bwasdetected bymass spectroscopy, Iwas not ableto detectthe remaining portionsofSP-B using thismethod.
250 350 450 550 650 750 850 M/Zamu
MS/MSspectrum ofSN-SP-B
172.0495 275.1224
I
o~-'.:'...:.~~~~~~~~~~-~~~~~.:!.-
50
MS/MSspectrum ofSN-SP-B
60 55 50 45
>40
i Ii
o~~~~~~~~~~~~~~'-!-, 50
Figure8.MS/MSspectraofSN-SP-Bsampleproducedusing MALDI-TOF mass spectroscopy.A) The mass/chargeratios ofthe fragment correspondingto histidine tag sequence islabeled.B)Peaks corresponding to thesequence ofN-terminalfragmentofSP-Barelabeled.Theamino acid sequcnces of these detectedfragments are shown in Table3.
Table3.MS/MS analysisconfinned thepresenceof twofragment swith the fromtheSN-SP- Bconstruct(indicated by gray highlighting).Red representsthe SP-Bsequenceand bluerepresentsthe fusionpart consisting ofthe sequenceofhistidine tag, SN and thelinker.[RG] isthe actionsite of thrombin.Trypsinpotential cleavage sitesare underlined.
SN-SP-Bexpectedaminoacidsequence
ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPE
ADGKMVNEALVRQGLAKVA YVYKPNNTHEQHLRKSEAQKKEKLNIWSED
CWLCRALIKRIQAM IPKGALAVAVAQ VCjiVVPL VAGGICQCLAERYSVlLLDTLLGjiMLPQLVCRL VLjiCSM A.[)ctectcdSequenceofthefusionpart
3.2. Investigatingdisruptionoj protein syn thesis ami proteolysisinduced bystress-activatedproteins
The electrophoresisand mass spectrosco pywork in thepreviou s section broughtup thepossibilitythat eitherSP-B maybeproducedbythe bacteria inatruncatedform,ormaybeproteolyzedduringits expression.To investigatethesepossibilities,cellcultures were collectedI hour, 3hours,5 hours and18 hours(overnight ) afterinduction by!PTG.Afterthecell disruption,Western blottingof thecellextractshowed bands atthe same molecular mass(-22kDa)for alltimepoints.Results showed thatthe expressedSN-SP-B did not changeat differenttimepointsafterinduction (Figure9).
Figure9.Western blot representation oftime coursecollectedculturesafter induction.a)I hour;b) 3hours;c) 5hours;d)18 hours (overnig ht)after inductio n.Protein concentrationwasnot equal insamples;the presenceand the size oftheband wereonlyinvestigated.
3.3.Investigating effectofcell disruption techniques011the expressed protein
Anotherpossibleexplanationforthelowmolecularweight observedforthe expressed protein waspeptidebreakagebymechanical forcesduring cell disruption.Althoughsonication usually doesnotbreak covalentpeptide bond s,thepossibledestructive effectofFrenchpress and sonication wereinvestigated (Figure 10).A combinationof these techniques were appliedtothe cell pellet;two timesFrench press and twotimes
sonication, one timeFrench press and two timessonication, twotimes Frenchpress, one timeFrench press, and fivetimesfreezeand thaw as a control. The detectedband in westernblot analysis wasidenticalinapparent size underall conditions(-22kDa) confirming that expressedSN-SP-Bwas notcleavedbycell disrupti onforces.
Figure 10.Westernblot analysisof100mlcell extracts subjec ted to different cell disrupti ontechniqu es.a)2times French pressand2times sonication;b)Itime Frenchpress and2times sonication;c) 2times French press;d)I time French press;ande)5 timesfreeze and thaw.Protein concentration wasnot adj ustedinsamples; the presenceand thesizeof the band only were investigated.Diffu sionofbandsisdue tosample overloading.
3.4. Optimizinggrowthcon ditions
lncreasing thelevels of SN-SP-1l expressionwasimportantboth for better conlirm ingth e sequence ofth e expressedprotein,as well asforfuture structuralstudies.Therefore 1 next worked to optimize bacterial growt hand protein expressionconditions. The typeof themedia andconcentrationof lPTG forinduction wereinvestigated (Table4).First, the total protein concentrationwasmeasured fo r 500mlcellculturesgrown inTil(Terr ific Broth)and LB media. Thecultureswere inducedbyI mM IPTG.The total proteinyield Protein concentration increasedsignificantlyin Tilwhichisa
A previousstudyshowed thatprotein expressio ncan beimproved by decreasing theconcentrationoflPTGusedto induceexpression (Romano,etal.2009).Iexamined the effectofIPTG concentrationonSN- SP-Bexpression bythetransformed cells. Protein contentwasmeasured after inducing LBcultures by a gradientoflPTGconcentrations.Results showed thatup to 0.4 mM IPTG,the totalprotein concentration is increas ing.Decreasesin protein concentrationwereevident at higher concentrationsoflPTG.Both 0.4mMand I mM IPTG were testedinTB media cultures.Total protein contentwas significantly higher forcultures inducted by 0.4mM lPTG comparedtoI mM IPTG.
Table 4.Optimizing !PTG concentrationto maximize protein express ion.
Total protein concentrationof sampleswithequalvolume,resultingfrom cellextractsof500 mlovernightcultureswas measured bythe Bradford assay.LB cultureswere inducedby four differentIPTG concentrat ions.TB media cultureswere inducedonlyby0.4mM and I mM[PTG.Xmeans theseconcentrationsof[PTGinTB were notinvestigated.
IPTG(mM) LB(mglm l) TB(mglml)
3.5.Production of 0no velSP-B peptide; Nstermin aldeletedSP-B peptid e
The difficulties in confirming the fullsequenceof the recombinantly expressed SN-SP-Bby mass spectroscopyarelikely related to the highlyhydrophobicnature o f SP-B.Parallel studieswithfragmentsof SP-B(seesection3 .9)indicatedtheN-term inaI7 residucsofSP -Bmightbe particularly problema tic.Evenif we couldconfirmthefullsequence,we wouldface similardiflicultiesinfurtherstructuralstudiesonthe sample.ln order to producea novelnear full-lengthSP-Bpeptide withoutaffecting its expectedhelical structure,seven amino acids fromN-termina lendof SP-B were removed by site directed mutagenesis(Figure II). Theresultinggene
had a histidinetag followed by SN (asthe fusion protein) andN-tenn inal deletedSP-B(Nd-SP-B).DNA sequencingofmultiple transformed clones con finnedthe successof mutation.
MHHHHHHA TSTKKLHKEPATLIKAIDGD TVKLMYKGQPMTFRLLLVDTPE TKHPKKGVEKYGP EASAFTKKMV ENAKKIEVEFDKGQRTDKYGRGLAYIY ADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQKK EKLNIWSED NADSGLGGGGGL VP[RGjPCWLCRA LlKRIQAMIPKGALAVAVAQVCRVVP LVAGGlCQCLAERYSVILLDTLLGRMLPQLVCRL VLRCSM
FigureII. The expectedaminoacid sequenceofN-tenni nal deleted SP-B peptide.Blue representsthesequence ofthe fusionprotein;staphylococcus nuclease (SN). Red is theexpectedsequenceforN-tenn inal deleted SP-B (- 7AA),[RGj is therecognitionsiteof thrombin .
3.6. Identifi cationofexpressed recombinantN-terminal deleted SP-B
Themutated plasmid containing SN-Nd-SP-Bwastransformed into theC43E.coli strain. Thecell extractfroman overnightinducedculture was purifiedon a cobalt column(Figure 12).Western blotanalysis identifiedtwobands-2Iand22kDawiththe histi dine tag.S incebandsare
very close they canbe res ultof the parti alredu ct ion of disulfide bondsin the protei n.Thefractionof protei nremai ningwithdisu llidebonds mo ve slowe r throu gh the gel than thereducedfractio nof protein.C43(DE3) are mutantsthat allowover-expressionof some globularand membran e protei ns unabl eto be expresse dat high-l evelsinthe parent stra in BL2I (D E3).
Cysteinesin theE.colicytoplasm(includi ngC43(DE3))are active lykept reducedbypathwaysinvolving thioredoxinredu ctase and gluta redoxi n.Th e disulfide bonddependent foldingofheterologo usproteins is improve din some strains(theOrigami strainsby Novagen)(Sorensen,H.P.&
Mortensen,K.K.,2005).In my work the repr esen tedband s are frompost celldisruptionsteps .After therupture,mostlikely disulli debondforma tio n happens.Howeverthemolecularmass for both band sislessthan expec ted forNd-SN -SP -B(-26kDa).Thesample waspurifiedthrou gh coba lt columnandwas digested by trypsinfor MALDl-TOF MS[genom ics and proteomics(GaP)facility,Memo rialUnivers ity]. Mass spec troscopycould detectfragment s from SN andthe linker part ofSN-Nd-SP-B(Figure 13 and TableS).
Figure12.Western blot analysis of the cell extract from anovernight induced culture encodingSN-Nd-SP-B .Bands representantigen-antibody reactionuponapplying anti-Histidineantibody. The histidinetag islocated at theN-terminusof SN-Nd-SP-B. Samples from differentstages of protein extraction wereloaded on thegela) supernatant aftercelldisrupti on and solubilisation(1%lauroyl sarcos ine) b) 30IIIofS N-Nd-SP-Bresultin gfrom the elutionstepof cobaltaffi nitychromatography(applying200mM imidazole)c) 60IIIof elutedSN-Nd-SP-Bd)flowthroughafterload ingthe sampleande) washafter loadingthe sample(75mM imidazole).Protein concentration wasnot adj ustedinsamples;the presence andthe sizeofthe band were investigated.
- - - - -
MS-MSspectrumofSN-Nd-SP-B
60 1026.561 ,60
50
~40 272.172
j ::
175.1187228.133810
I
J.llljli1.1.l'W"i1'JlJd.LI.,.~i."",:',L,,' ,,
""..,[ , .I
0 .t.
50 550 1050 1550 2050
A
MS-MSspectrumofSN-Nd-SP-B
20 84.0776 18 16
1007.532
14 I
.~ 12 1ii1O
JI I
E
8 6J
2 I :1, ,I lLlI_JLJJ
0
50 250 450 650 850 1050
B M/Zamu
Figure13.MS-MS spectraresultingfrom MALDI-TO Fmass spectrosco py of SN- Nd-SP-I3 trypsindigested sample. A) The mass/char geratio of a fragmentof SN islabeled.B)Peaks corresponding tothe sequenceof SN and the linkerare labeled. Theaminoacidsequencesof the detected fragmentsareshowninTable
Table5.MS/MSanalysisof SN-SP-Btrypsin digested sampleidentifiedtwo fragmentswith the expectedsequence(indicate d by grayhighlighting).No fragmentwas detectedfrom Nd-S P-B. Red represents the SP-Bsequenceand blue representsthe fusion part consistingofthe sequenceofhistidine tag,SN and the linker.[RG)istherecognition siteof thrombin. Trypsin potentialcleavage
SN-Nd-SP-Bexpectedaminoacid sequence
MHHHHHHATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLL VDTPE KHPKKGVEKYGPEASAFTKKMVENA_
DGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQKKEK
AGGICQCLAERYSVILLDTLLGRMLPQLVCRL VLRCSM DetectedSeq uenceof thefusionpart
DetectedSeq uence ofNd-S P-B
3.7.ProductionandcharacterizationofNsterm inalinsertionsequence (SP-HpJ
Thefirst7residuesofSP-B(the insertion sequence)wereproduced by chemicalsynthesisfor structuralstudies.SP-BI_7was synthesized by Fmocchemistry(section2.6).The peptide waspurifiedbyusing reverse- phaseHPLC.TheHPLC wasequippedwithareverse-phaseDYNAMAX C8preparatorycolumn(Figure 14).The purifiedpeptide was collectedfor furtherstudies bymass spectroscopy.The mass andsequenceof thepeptide wereconfi rmed throughMALDI-TOF MS[Genomics and Proteomi cs (GAP)facility,MemorialUniversity].
Figure 14.HPLC spectrumofS P-BI_7eluted withan increasing acetonitrile gradient.Sampleswere runthrough apreparatory reverse-phase HPLC
chromatographerequipped withaOYNAMAX C-8 column.The strongest [highestabsorbancein mill i-absorbanceunits{mAUlat 215 nm] peak{-19 min) wascollected tor sequencing bymass spectroscopy.The strong peakat the beginnin g of thespectrum belong sto water.
Circular dichroism studies werecarried out toassess secondary structural characteristicsof SP-B1•7•CO spectraof 2 mM Super-Mini-B wererecorded at25°Cand pH5inaqueoussolution with 40%HFIP (plus 50%H20 and 10%020),aswell asmembrane-mimeticenvironments (90%
~hO,10%020 ,0.2mM OSS,and 300 mM deuterated SOS).Efforts to solubilize thepeptide either in40%HFIPor SOS buffer werenot successful;mostof thesample wasinsoluble and precipitated.Second ary structural feature s of the soluble portion were calculated from the CD spectra using COProsoftware (http://lamar.col state.edul-ssreeraml COPro ) andCONT IN/LL analysismethod (Sreeramaand Woody,1993;2004).The results forSP-BI_7in 40%HFIP and SOS buffer were simi lar;the curvesdid nothavethetypicalpatterntora helicalconf ormat ion(Figure15).The calculatedsecondary structural contentwere similar for HFIPand SOS containingsamples; mostlyunordered structures (45.8%in HFIP,46.1% in SOS)and~-structures(27.3%in HFIP,26.7% in SOS).A verysmall portionwasdetectedtor a-helicalstructures {0.3% and-1.5%31O-helixin 40%HFIP andSOS,31o-helix is aright-handedhelical structure), the
I
~-100 0peptid eretainsthe same cont ent of 13%polyprol ineIIstruc ture in 40%
HFIPandSOS(Table6).
N-Term Peptide
- HFIP
- S OS
-2 0001+90 - - - - . -2.--10----.22,-0-2.--30---,24,-0 - - ,----,
Figure IS.Far-U VCOspectrumof 2mMN-terminalSP-B peptide dissolved in40%HFIP (plus50%H20and 10%020 ) at pH5 (red)as well as in thepresence of 300mMSOSNMR buffer containing90%H20,10%
020,0.2mMOSS,and 300mMdeuterated SOS(green).Allspectra were takenusing a I mm path-lengthquartzcuvettefrom 193nm to 260nm at
Table6.Secondarystructural contentof 2mM SP-B1•7in the presence of 300 mM SDSNMR buffer,and 40%HFIPNMR bufferat pH 5 and 25°C.
Structura lcontent percentageswere calculated fromCDspectra data using Woodyand co-workersmethod(Sreeramaand Woody1993;2000;2004).
3.8.SolutionIII/clearmagnetic resonancespectroscopy (NMR) preliminary structuralcharacterizationof N-terminal insertionpeptide
The structural characteristicsofSP-B1•7werestudied bysolution NMR experiments.The 2D TOCSYand NOESYare thestandard experiment sto identifyinteractionsbetweenspinsystems.
2DNMR experiment swith SP-B'.7in SDSmicellescoulddetect only a few peakswithlow intensities.Even though between spinsystems SP-B'.7isasmallpeptide,thenumber of observedpeaks wasstillfewer than expected.This couldbe becausethepeptideformslarge aggregated particleswhich do not give riseto solutionNMR peaks(Figure 16).
Aswasmentionedinsection2.7,ina NMRspectrumeach peakis associated with a specific magnetic (V, spin) nucleusin theinvestigated molecule.Ina 2DNMRspectrum,eachcross-peak is associatedwith2 nuclei.Inorder to assign thepeaksin the NMR spectraofSl-- Bj.-,firstthe TOCSYwas employed to indicate whichpeaks were in the samespin systemand hence inthesameaminoacid residue. Thespinsystemswere then assigned to anaminoacidtypebasedon comparisontotherandom coil values(Wuthrich,1986). The majority of thepeaksin theTOCSYspectrum were assignedwithin±O.7ppmoff theexpected proton resonance frequencies. [havedesignatedprotonnucleiinorderofproximit yto backbone HNnucleus,HA,liB,HC....(Table7). All of the 7residues (SP- BI•7)were assigned,however someoftheir protons did notproduce any observa blecorrelation peaksin ordertobe assigned.For prolines,because ofthcresonancesimilarity itwasnotpossibleto assign theirresonancesto particular prolineswithin the sequence.The remainingunassignedpeaks likely originatedfrom peptid eimpurities. Changesin intensity of peaks wereused to assessthe positionofS P-B1.7in the micelle. 16-doxy l-stea ric acid (l6-DSA)was added tothepeptide/micelle sample,DSA is a paramagneticdetergent-solubl ereagent.Theproximitytothisreagentleads to amarkedintensity reductionof allNMRsignalsof nearb yprotons (Hilty etaI.2004).
Alinewidthbroadening andthus aheight reductionof signalswere observedfor theentire spectrum.Thelevel of protonintensity reduction indicatesthe proximityof protongroupstothe centerofmicelle.The intensityreductionof peakswasmonitored inTOCSY spectra,and then peakintensitieswere usedto preparea plot(Figure 17). Dataanalysis showedasubstantialdecreaseinpeakintensitiesanddisappearanceofpeaks correspond ingto residuesIand2suggestingthcscrcsid uesarc dceply insertedin the micelle.Howeverunexpectedly several peaks showed increaseinintensity.
Table7.'H Chemicalshifts of theN-tennina linsertion sequenceofSP-B from the TOCSY experiment on SP-B'_7.HA,lID,...respectivel y stand for proton nuclei inorder ofproximitytobackboneHNnucieus.
HB (p pm)(ppm) (ppm)
Not 4.518 2.739 3.084 found
HC (ppm) 7.292
HD HE HF HG
(ppm) (ppm) (ppm) (ppm) 7.672 6.8657.3757.277
3.847 1.7812.122 1.292 1.545 4.382 1.771 2.283 1.941 1.778 4.531 2.0382.141 1.544 1.160
3.6593.176 --- 3.474 3.236 3.788 3.449
Not found Not found
Not 4.397 found
0.9940.9981.134 Not found
7 6 8 1Hfrequency(ppm)
0 0
0
0"
0',
4 4
•
8
~
lHfrequency (ppm)Figur e16.(A)20 TOCSY spectraof 2 mM SP-B1•7in300 mM SOS, at pH 5 and 45°C.TheHN-HXcorrelationregionispoint edout throu ghthe panelcoming off the spectra. HXcould beany type of proton nuclei except HN.(B)at pH5 and 45°C after additionof0.6mM 16-0SA.128 scans were recordedfor each experimentat 80 msmixingtime.
DSA/NoDSA2D TOCSY cross-peaksintensityratio
Figure17. Effectof0.6mM 16-0SAonNvterminalinsertion peptideof SP- B.Theratio of20 TOCSY cross-peak intensitywith and without 16-0 SAis plotted.X axisrepresentsthe aminoacidand correspondin gproton groups of eachcorrelationpeak.HA.HB•...respectively stand forprotonnucleiin orde rof proximityto backboneHNnucleus. Note that it wasnotpossibleto assign the prolineresonance stoparticul arproline swithinthe sequence.
