consists68vvv

[8]HOMONUCLEARHARTMANN--HAHNEXPERIMENTS151[8]HomonuclearHartmann-HahnExperimentsByADBAXIntroductionTheprinciplesandapplicationsofhomonuclearcorrelationexperi-mentshavebeendiscussedbyMarkley.
1Inthischapteroneofthesetechniques,knownasHOHAHAorTOCSY,willbediscussedinmoredetail,withparticularemphasisontheexperimentaldetailsrequiredforrecordingoptimalspectraonbiologicalsamples.
Totalcorrelationspec-troscopy(TOCSY)wasfirstproposedbyBraunschweilerandErnst.
2ThisexperimenthasparticularpotentialforproteinNMRstudiessinceitper-mitscorrelationofallprotonswithinascalarcouplingnetwork.
Hence,acompletesubspectrumcanbeobtainedforeveryaminoacid,makingresonanceassignmentsconsiderablyeasier.
However,theTOCSYexper-imentnevergainedmuchpopularity,mainlybecauseofthelimitedbandwidththatcouldbecoveredwiththemixingschemesusedintheoriginalpulsesequence.
Weaccidentallyrediscoveredthistypeofmagnetizationtransferwhenanalyzingartifacts3occurringinrotatingframeNOE(ROESY4,5)spectra.
ItwasshownthattheseartifactsarecausedbyahomonuclearHartmann-Hahn(HOHAHA)effectthatcouldbedescribedquantitativelybymathematicalexpressionsderivedfortheheteronuclearcasebyMiillerandErnst6andChingasetal.
7MagnetizationtransferviatheHartmann-Hahneffectoccursonlywhenthedifferenceintheabso-lutemagnitudesoftheeffectiverffieldsexperiencedbytwocoupledspinsissmallerthanthescalarinteractionbetweenthetwospins.
Theeffectiverffieldisthevectorsumoftheresonanceoffset,8,andtheappliedrffield.
Itsmagnitudeisthereforestronglydependentontheresonanceoffset.
ByconsideringthespuriousmagnetizationtransferintheROESYij.
L.
Markley,thisvolume[2].
2L.
BraunschweilerandR.
R.
Ernst,J.
Magn.
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53,521(1983).
3A.
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63,207(1985).
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Bothner-By,R.
L.
Stephens,J,T.
Lee,C.
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Jeanloz,J.
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1116,811(1984).
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FarmerII,thisvolume[11].
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Ernst,Mol.
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38,963(1979).
7G.
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Chingas,A.
N.
Garroway,R.
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Bertrand,andW.
B.
Moniz,J.
Chem.
Phys.
74,127(1981).
Copyright1989byAcademicPress,Inc.
METHODSINENZYMOLOGY,VOL.
176Allrightsofreproductioninanyformreserved.
152ADVANCEDTECHNIQUES[8]experimentasacaseofhomonuclearHartmann-Hahncross-polariza-tion,itwasrelativelystraightforwardtodevelopmethodsthatwouldovercometheeffectsofrfoffset.
Aseriesofdifferentmixingschemesisnowavailableandtheiradvantagesanddisadvantageswillbediscussed.
Aswillbeshown,theHOHAHAexperimentsareveryefficientatestab-lishingdirect(COSY8)andindirect(RELAY9)typesofspin-spinconnec-tivities.
OfparticularimportanceforthestudyofproteinsisthefactthattheefficiencyofHOHAHAmagnetizationtransferislesssensitivetoshorttransverserelaxationtimesthanthealternativeCOSYandRELAYtypeexperiments.
Aswillbedemonstratedhere,methodsforrecordingtheHOHAHAspectrainH20solutionareespeciallyusefulforconnect-ingtheside-chainprotonswiththeusuallybetterresolvedamidereso-nances.
Atpresence,mostcommercialspectrometersrequiresomespe-cialhardwareforproducingtherffieldsthathavetobegeneratedduringthemixingperiod.
Therefore,abriefdiscussionofthehardwarerequire-mentsoftheHOHAHAexperimentisalsoincluded.
PrinciplesofHomonuclearHartmann-HahnSpectroscopyAdetailedtheoreticalanalysisofHartmann-Hahncross-polarizationisbeyondthescopeofthischapter.
Forbackgroundinformationonhet-eronuclearHartmann-Hahncross-polarizationinliquidsthereaderisreferredtothepapersbyM011erandErnst6andChingasetal.
7Foranalo-gousdescriptionsofthehomonuclearcase,thereadercouldconsultavarietyofpapers.
2,3,1°-~4Here,theprincipleswillbebrieflyreiteratedtoprovidesomeinsightintoselectingtheoptimalparametersforaparticularexperiment.
AspointedoutbyBraunschweilerandErnst,themagnetizationofaspin,I,underisotropicmixingconditionsisperiodicallyconvertedintomagnetizationofasecondspin,S,providedthatIandSarescalarcou-sK.
Nagayama,A.
Kumar,K.
Wuethrich,andR.
R.
Ernst,J.
Magn.
Reson.
40,321(1980);A.
BaxandR.
Freeman,ibid.
44,542(1981).
9G.
Eich,G.
Bodenhausen,andR.
R.
Ernst,J.
Am.
Chem.
Soc.
104,3732(1982).
i0A.
BaxandD.
G.
Davis,in"AdvancedMagneticResonanceTechniquesinSystemsofHighMolecularComplexity"(N.
NiccolaiandG.
Valensin,eds.
),pp.
21-48.
Birkhaeuser,Basel,1986.
Nj.
S.
Waugh,J.
Magn.
Reson.
68,189(1986).
12R.
BazzoandJ.
Boyd,J.
Magn.
Reson.
75,452(1987).
13R.
R.
Ernst,G.
Bodenhausen,andA.
Wokaun,"PrinciplesofNuclearMagneticReso-nanceinOneandTwoDimensions,"p.
444.
OxfordUniv.
Press(Clarendon),LondonandNewYork,1987.
14A.
Bax,J.
Magn.
Reson.
77,134(1988).
[8]HOMONUCLEARHARTMANN--HAHNEXPERIMENTS153pied.
Mathematically,thisisdescribedbytheexpressionJ~'ISIx>Ix[l+cos(27rJ~-)]/2+Sx[1-COS(27rJT)]/2+(IySz-IzSy)sin(27rJT)(1)where~"isthedurationofthemixingperiod.
IxandSxdenotethexcomponentsoftransverseIandSspinmagnetization,andsimilarlytheindicesyandzrefertotheyandzcomponentsofmagnetizationintheregularrotatingframe.
ThisexpressionshowshowthexcomponentofIspinmagnetizationisconvertedperiodically(withperiod1/J)intothexcomponentofSspinmagnetization.
Similarly,duringisotropicmixing,lyisperiodicallyinterconvertedwithSyandlikewiseIzwithSz.
Ifthemag-netizationtransfertakesplaceduringthemixingperiodofatwo-dimen-sional(2D)experiment(Fig.
1),thisgeneratesoff-diagonalcross-peaksinthe2Dspectrum.
Equation(1)showsthatfortwocoupledspinsallIspinmagnetizationcanbetransferredtoS(intheabsenceofrelaxation)ifthedurationofthemixingperiod,~-,issetto1/(2J).
Thediagonalpeaksinthiscasewoulddisappearfromthe2Dspectrumandallintensitywouldbepresentinthecross-peaks.
Forsystemsconsistingofmorethantwospins,ananalyticalexpressionanalogoustoEq.
(1)forthetransferofmagnetizationbecomesmuchmorecomplicated,~5:6andcompletetrans-ferofmagnetizationfromthediagonaltothecross-peaksisgenerallynotpossible.
Moreover,inproteinstudiesshortrelaxationtimesprohibittheuseoflongmixingperiodsneededfortransferringthemaximalamountofmagnetizationtothecrosspeaks.
Equation(1)indicatesthatin-phaseIspinmagnetization(Ix)canbetransferredtoin-phaseSspinmagnetization.
Thisisverysimilarto,forexample,theNOESYexperimentinthatonecanrecordthespectrumintheabsorptionmode,withallcross-peakshavingthesamesignasdiago-nalpeaks.
However,morecarefulinspectionofEq.
(1)showsthatIxmagnetizationcanalsobetransferredinto]ySzand[zSyterms.
Theselattertermsdenoteantiphasemagnetizationcomponents;forexample,I~SrreferstoSspintransversemagnetizationalongthe-yaxis,i.
e.
,themagnetizationvectorthatcorrespondstotheSspindoubletcomponentwithspinIintheaspinstatepointsalongtheyaxisandtheseconddoubletcomponentpointsalongthe-yaxis.
BecausethelinewidthsinproteinspectraoftenarelargerelativetotheJcoupling,theoverlappingantiphasemultipletcomponentslargelycanceloneanotherandthespec-trumstillappearstobeabsorptive.
Glycineresiduescanbeanexceptiontothisrulebecausetheirdoubletcomponentsoftenarepartiallyresolved15N.
Chandrakumar,J.
Magn.
Reson.
71,322(1987).
16N.
ChandrakumarandS.
Subramanian,J.
Magn.
Reson.
62,346(1985).
154ADVANCEDTECHNIQUES[8]atli°,btli"ctli°,dt1SPINLOCKy!
/~/~/~AAAA.
Vvvvv-180y180yIHJllllllllll-'1SLyMLEV17SLy~i0t1SLyIWALTZ17ySLy[FIG.
1.
Pulseschemesfor2DhomonuclearHartmann-HahnandTOCSYexperiments.
(a)Usingaspin-lockfieldofconstantphase,requiringexcessivelystrongrffieldsforcross-polarizationoverawidebandwidth,butpossiblyusefulforres:rictingmagnetizationtrans-fertospinsthatarecloseinchemicalshift.
(b)TheoriginalTOCSYexperiment,2usingaseriesofcloselyspaced180°pulsestoaccomplishmagnetizationtransfer.
Theshortestpossible180°pulsewidth(orcomposite180°pulsest3)shouldbeusedandpulsespacingshouldbelessthan1/(2SW),whereSWisthespectralwidthinhertz.
(c)Magnetizationtransferusingaphase-alternatedspin-lockscheme.
(d)MixingusinganMLEV17cycle,precededandfollowedbytrimpulses(typically1-2mseceach).
TheMLEV17rmixingconsistsofanintegralnumberofrepetitionsofthefollowingsequence:ABBABBAABAABAABB60y,whereA=90x180y90xandB=90_x180_y90_x.
(e)MixingusingtheWALTZ17ycycle,consistingofanintegralnumberofrepetitionsofABBA-ay,whereA=270_x360x180_x270x90_x1801360_x180~270_xandB=270x360_x180x270_x90x180_x360x180~270x;theflipangleaisadjustedbetween0and90°.
Thephasecyclingusedis~b=x,-xandAcq=x,-xforTPPItypeexPerimentsand~b=x,y,-x,-yandAcq=x,x,-x,-xforhypercomplexdataacquisition.
Inaddition,CYCLOPSmaybeaddedbyincrementingallphases(~b,trimpulses,spinlockorMLEVorWALTZandAcq)by90°aftercompletionoftheshortphasecycle.
(largecouplingwithnopassivespinsiftheNHprotonhasbeenex-changedfor2H)andthephasedistortionofthecross-peakbetweenthegeminalprotonsthencanbeobserved.
Equation(1)isonlyvalidifallchemicalshiftandrftermsoftheHamiltonianhaveidenticalvaluesforeachofthetwospins.
Forexample,[8]HOMONUCLEARHARTMANN-HAHNEXPERIMENTS155iftheJ-coupledspinsIandShaveidenticalchemicalshifts,periodicoscillationofmagnetizationbetweenspinsIandSoccurswithoutanyrffieldbeingpresent(infinitelystrongcouplingcase).
Ingeneral,spinsIandShavedifferentchemicalshiftsandthepurposeoftherfirradiationistoeliminatethedifferenceinthechemicalshiftsbetweenthetwoprotons.
Thisthenwouldcreateasituationwherethespinswouldbecomeinfi-nitelystronglycoupled,resultinginperiodictransferofmagnetizationbetweenthetwocoupledspins.
Onesimplewaytoaccomplishthisistheapplicationofastrongrffield(Fig.
la).
Wewillconsidertheeffectiverffieldsexperiencedbythetwocoupledspins.
Eacheffectiverffieldcorre-spondstothevectorsumoftheappliedrffield(alongtheyaxis)andtheresonanceoffsetvector(alongthezaxis)forthatparticularspin(Fig.
2).
Themagnitudeoftheeffectiverffield,yell,foraparticularspinwithresonanceoffset8fromthecarrieristhengivenbyVeil=(82+v2)1/2(2)wherevisthenominalrffieldstrength(infrequencyunits).
Forcaseswherethefffieldstrengthismuchlargerthantheoffset(v>>8)thedifference,A,ineffectivefieldstrengthsfortwospinsIandSisA=peffI--/.
,eftS2)/21.
,(3)AconditionforHartmann-HahntransferisthatA~J.
AscanbeseenfromEq.
(3),onewaytoobtainthisistouseaverystrongrffield.
Alternatively,thecarriercanbeputexactlyhalfwaybetweenthetwoZ161.
.
.
.
.
~Veff1I~Veff'/xFIG.
2.
Vectordiagramoftheorientationsoftheeffectivefffieldsintherotatingframe,experiencedbytwospinswithoffsets81and82,whenanfffieldofstrengthvisappliedalongtheyaxis.
156ADVANCEDTECHNIQUES[8]resonances,suchthat8~=-Ss.
AsecondpointworthnotingisthattheeffectiveJcouplingduringspin-lockconditionsisreducedbecausethetwoeffectivefieldspointindifferentdirections.
Thisslightlyslowsdowntherateofmagnetizationtransferbetweencoupledspins.
~1,12,~4Inprac-tice,themethodofFig.
laisnotsuitableforwide-bandcross-polariza-tion:anrffieldofseveralmegahertzwouldbeneededtocovera10-ppmbandwidthat500-MHzJHfrequency!
Inaddition,transverseNOEwouldalsogiverisetointensecross-peakswhichmightcanceltheHartmann-Hahntransferswhichareofoppositesign.
ThesameistrueforthemixingschemeusedintheoriginalTOCSYexperiment(Fig.
lb).
Althoughthepowerrequiredforwide-bandhomonuclearcross-polarizationissignifi-cantlyreducedwiththismethodrelativetocontinuousirradiation,trans-verseNOE'sarealsopresentandthebandwidththatcanbecoveredeffectivelyisstillquitenarrow.
MixingBasedonCompositePulseDecouplingSchemesEffectivemethodsforwide-bandcross-polarizationinproteinsmustsatisfythreecriteria:(1)theeffectsoftransverseNOE'smustbemini-mized;(2)minimalrfpowermustbeusedduringthecross-polarizationtoavoidsampleheating;and(3)theymustbeeasytouseinaroutinefashion.
Below,twosequencesthatsatisfythesecriteriawillbediscussedbriefly.
ArgumentspresentedbyWaughonthetheoryofspindecoupling~7suggestedtousthatsequenceseffectiveatbroad-bandheteronuclearde-couplingmightbeusefulforobtainingwide-bandhomonuclearcross-polarizationatlowrfpower.
ThefirstimprovementwemadeontheschemeofFig.
lawastomodulatethephaseofthespin-lockfield(Fig.
lc),theanalogofsquare-waveheteronucleardecoupling.
18MuchmoreeffectivedecouplingschemeshavebeendevelopedbyFreeman,Levitt,andco-workers.
~9,2°Inafirstattempt,wetriedtousetheirMLEV16scheme21forwide-bandhomonuclearcross-polarization.
TheMLEV16compositepulsesequenceconsistsofalargenumberof90and180°pulsesthatarephaseshiftedrelativetooneanother,asdescribedinthecaptionofFig.
1.
Thefirstproblemencounteredwasthatthisschemepreservesboththexandtheycomponentoftransversemagnetizationpresentattheendoftheevolutionperiod.
However,therelaxationratesofthesetwo17j.
S.
Waugh,J.
Magn.
Reson.
50,30(1982).
isj.
B.
GrutznerandA.
E.
Santini,J.
Magn.
Reson.
19,178(1975).
19T.
A.
Frenkiel,M.
H.
Levitt,andR.
Freeman,Adv.
Magn.
Reson.
11,47(1983).
2oA.
J.
ShakaandJ.
Keeler,Prog.
NMRSpectrosc.
19,47(1987).
21M.
H.
Levitt,R.
Freeman,andT.
A.
Frenkiel,J.
Magn.
Reson.
47,328(1982).
[8]HOMONUCLEARHARTMANN--HAHNEXPERIMENTS157componentsdifferfromoneanotherduringtheMLEV16irradiation(videinfra),resultinginquadratureartifactsandphasedistortions.
Asdis-cussedlater,trimpulsesbeforeandafterthemixingperiodcanbeusedtoeliminateeitherthexortheycomponent.
TheMLEV16schemeissup-posedtoaccomplishisotropicmixingoftheprotonsoverawidebandwidth,whichmeansthatthemagnetizationvectorofanyisolatedspinintherotatingframeendsupinitsstartingorientationafterasingleMLEV16pulsecyclehasbeencompleted.
AscalculatedbyWaugh,HevenintheorytheMLEV16sequenceisnotquiteperfectatdoingthis.
Inpractice,smallerrorsinphaseshiftingandpulsewidthaggravatethisimperfection.
Soitcanhappenthatymagnetizationpresentattheendoftheevolutionperiodfinishesalongthezaxisattheendofthemixingperiod(aftermanyMLEV16cycles),andremainsundetectedduringthet2periodofthe2Dexperiment.
Thisthenresultsinphaseandamplitudedistortionsinthe2Dspectrum.
Oursolutiontothisproblemwastoaddaseventeenthpulse(60y)whichpreventstheyspinmagnetizationfromrotatingawayfromtheyaxisduringsubsequentMLEV16cycles.
NotethattheadditionoftheseventeenthpulseseverelyreducesthebandwidthoverwhichthisMLEV17sequence22providesefficienthomonuclearcross-polarization(orheteronucleardecoupling).
However,itsbeneficialeffectsaretoensurethatpure-phasespectrafreeofanomalousamplitudedistortionscanberecorded.
Italsocanbeshownthattheadditionofthisseventeenthpulsemakesthemixingnonisotropic,whicheliminatesmag-netization(sensitivity)lossthatotherwisewouldbecausedbythegenera-tionofmultiple-quantumcoherence.
H.
23Asmentionedearlier,inprincipleboththexandtheycomponentsoftransversemagnetizationarepreservedduringthemixingperiod.
Theundesirablexcomponentcanbeeliminatedbytheapplicationofso-calledtrimpulsesbeforeandafterthemixingperiod.
Becauseofthesevereinhomogeneityoftherffield,anymagnetizationnotparalleltotheeffec-tivefieldvector(theyaxisinFig.
ld)israpidlydefocused.
Theeffectofanynondefocusedxmagnetizationthatcangiverisetoaweakanti-diagonalinthe2Dspectrumcanbeeliminatedbycoaddingtheresultsoftwo2Dexperimentsrecordedunderidenticalconditionsexceptthatthesecondexperimentusestrimpulsesthatare90°longerthanthefirstexperiment.
23UseoftheMLEV17sequenceforproteinstructuredeterminationhasbeenpioneeredbyClore,Gronenborn,andco-workers,24anditisnow22A.
BaxandD.
G.
Davis,J.
Magn.
Reson.
65,355(1985).
23A.
Bax,lsr.
J.
Chem.
28,inpress(1989).
24G.
M.
Clore,S.
R.
Martin,andA.
M.
Gronenborn,J.
Mol.
Biol.
191,553(1986).
158ADVANCEDTECHNIQUES[8]usedroutinelyinanumberoflaboratories.
TheMLEV17sequenceisquiteforgiving;thesequenceisnotparticularlysensitivetothecalibrationofhardwarerfphaseshiftsorbalancingoftheamplitudesofthephase-shiftedpulses.
Exactcalibrationofthepulsewidthsalsoisnotverycritical.
Foreffectivecross-polarizationovera10-ppmbandwidthat500MHz(assumingtheworstcaseofanNHprotonremovedby5ppmfromthecarrierandC~Honresonance,withaJcouplingof4Hz),anrffieldstrengthofaboutl0kHzisneeded.
Typicallythisrequiresabout3-to5-Wrfpower.
Evenifthispowerisappliedforrelativelyshortmixingperioddurationsof30-60msec,thiscanresultinsignificantsampleheat-ingeffects.
Theinhomogeneoussampleheatingmaycausedeteriorationofthelocksignalandanincreaseintlnoise.
MixingbasedontheWALTZ1625decouplingcyclecanbeslightlymoreefficientthantheMLEV17scheme,providingareductioninrfpowerbyuptoabout40%.
However,ourcomputersimulationsandexperimentalresultssuggestthatthistypeofmixingismorecriticalwithrespecttoexactbalancingoftheamplitudesofthetwophase-shiftedrftransmitchannels.
OnsomeofthenewertypesofNMRinstruments(whichoftenusedigitalrfphaseshifting)thispresentsnoparticularprob-lemandtheWALTZ-basedmixingscheme(Fig.
le)isthenpreferableovertheMLEV17scheme.
ForsimilarreasonsasfortheMLEV17scheme,aseventeenthpulsemaybeaddedattheendofeachWALTZ16cycle.
Inpractice,onourBrukerAM600spectrometer,alengthof30-60°forthisseventeenthpulseissufficient.
Todeterminewhatdurationisneededfortheseventeenthpulse,thefollowingsimpleexperimentshouldbeconducted.
Useacompoundwithwell-resolvedresonancesspreadoveratleast8ppm.
Recordthespectrumthatcorrespondstothefirstincrementofthe2Dexperiment.
Firstuseashortmixingtime(oneWALTZ17cycle)followedbya3-msectrimpulseanddeterminewhatphaseparametersareneededforphasingthe1Dspectrumtotheabsorp-tionmode.
Next,usinga75-msecmixingperiod,removethefinaltrimpulse,andreducethewidthoftheseventeenthpulsedownfrom180°untilphasedistortionsbecomeapparentinthespectrum(whenphasedwiththeparametersfortheshortmixingperiodwithtrimpulse).
Theseventeenthpulseshouldbesettotheminimalwidthforwhichthespectrumstillhasanabsorptionmode(-15°)appearance.
AllspectrashowninthischapterhavebeenrecordedwiththeschemeofFig.
le,usinga60°flipanglefortheseventeenthpulse.
ReducingRelaxationduringMixingIntheschemeofFig.
la,thespin-lockedmagnetizationdecayswithatimeconstantTip,whichforproteinsisidenticaltoT2.
Intheschemeof[8]HOMONUCLEARHARTMANN--HAHNEXPERIMENTS159Fig.
ld,theymagnetizationpresentatthestartoftheMLEVI7sequencegoesthroughatrajectorywhereitspendsone-halfitstimealongthezaxis(relaxingwithTI)andone-halfitstimeinthetransverseplanewhereitrelaxeswithT2.
Becauseformacromolecules,1HTIvaluestypicallyaremuchlargerthanT2values,themagnetizationdecayisreducedbyalmostafactoroftworelativetoSchemesa-cinFig.
1.
22Similarly,duringtheWALTZmixingschemeofFig.
le,magnetiza-tionperpendiculartotheaxisalongwhichtheWALTZpulsesareapplied(thexaxisinFig.
le)relaxesbyalmostafactoroftwomoreslowlyrelativetomagnetizationparalleltothataxis.
Notethattheadvantageinrelaxationonlyappliestoin-phasespin-lockedmagnetization;theanti-phaseIySzandIzSyterms,essentialintheHOHAHAprocess,arenotreducedinrelaxationrate.
InterferencefromNOEEffectsAsmentionedearlier,transverseNOEeffectscaninterferewiththeHartmann-Hahneffects.
Since,underspin-lockedconditions,theNOEeffectisalwayspositive,NOEcross-peaksareofoppositesigntothediagonal.
DuringtheMLEVandWALTZsequences,however,the"spin-locked"magnetizationspendshalfitstimealongthezaxis,wheretheNOEisnegativeformacromolecules.
Althoughthespin-lockedNOEeffectislargerthantheregularNOEeffect,thefactthattheyareofoppositesignstronglyreducesthenetNOEeffect.
Thisminimizesreduc-tionofHOHAHAcross-peakintensitiesincaseswherebothHOHAHAandNOEeffectsarepresent.
NotethatfortheWALTZsequencetheNOEeffectswouldpersistifthephaseoftheWALTZpulseswerechangedby90°withrespecttothetrimpulses.
ApplicationsofHOHAHAtoPeptidesandProteinsThepoweroftheHOHAHAexperimenttosolvespectralassignmentproblems(cf.
Basus[7],Vol.
177,thisseries)isprobablymostclearlydemonstratedforpeptidesandproteinsbycorrelatingamideprotonswithC~andside-chainprotons.
Asanexample,Fig.
3showsthecomplete2Dcorrelationspectrumfortheantimicrobialpeptide,magainin2(23aminoacids),z6Thespectrumwasrecordedinamixtureoftrifluoroethanol(TFE)andH20(25/75,v/v)usingSchemeeofFig.
l,withpresaturationoftheH20resonancepriortotheevolutionperiod.
Becauseoftheacidicsampleconditionsused(pH4priortoadditionofTFE)theamideprotonexchangeissufficientlyslowthatpresaturationdoesnotobliterateanyof25A.
J.
Shaka,J.
Keeler,T.
A.
Frenkiel,andR.
Freeman,J.
Magn.
Reson.
52,335(1983).
160ADVANCEDTECHNIQUES[8]+,ei.
|°eOeeto!
eoi!
0~8:.
v;.
,+0oo+e,o.
.
.
.
q,+C2H/C4HQI/C~H/C~H24',"'1:.
.
,q.
,,,o,"]'.
0~.
m~oe#.
,,.
~7,.
~.
,.
40O0(I~O#""I:/k::it:|~!
oil,.
'"3|~/1414014'"t+'dP"~''":4<"g"Qt+,,.
°"F,6~7ee.
oeII,+eql|oooIIiii11141111tlq~tl°°"41-~0tl.
ooiFIG.
3.
Two-dimensionalHOHAHAspectrumofthepeptidemagainin2,15mgin0.
375mlH20/0.
125mltrifluoroethanol,27°.
Thespectrumwasrecordedat600MHz,usingaWALTZ17mixingsequencewitha=60°.
The90°pulsewidthwas35/~sec,durationoftheWALTZirradiationwas75msec,andthetrimpulsedurationwas2msec.
PresaturationoftheH20resonancewasusedduringtherecyclingdelay.
DatawereacquiredintheTPPImodeandthesizeoftheacquireddatamatrixwas700x2048,correspondingtoacquisitiontimesof42and120msecinthet]andt2dimension,respectively.
Eightscans,precededbytwodummyscans,wererecordedperttvalue,andthetotalmeasuringtimewas3.
5hr.
Zerofillingwasusedtoyieldadigitalresolutionof4Hzinbothdimensions.
[8]HOMONUCLEARHARTMANN-HAHNEXPERIMENTS161--002o--oI000Ala-gF21.
.
.
.
,--O0__t,+-,,0e82--Gly-13Ala-15lys-4--00++07.
+'~'Y~::L~--~--oVa'-1+l'l~-200Q--0c0--oPPu43v~21FIG.
4.
ExpansionoftheboxedregionofFig.
3,showingtheamidetoside-chainconnec-tivities.
WiththeexceptionsofGly-1andIie-2,foreachamideprotonnearlyallside-chainresonancesconnecteddirectlyorindirectlyviascalarcouplingstotheC~-Hresonanceareobserved,despiterelativelysmallNH-C~HJcouplings(ahelix).
theexchangeablepeptideprotons.
Undertheseconditionsthepeptideadoptsana-helicalconformation,27andconsequentlytheNH-C~HJcouplingsarerelativelysmall(4-5Hz).
Nevertheless,forthemixingtimeusedinthisexperiment(79msec)alargeproportionoftheamidemagneti-zationhasbeentransferredtothealiphaticprotons.
AnexpansionoftheboxedregionofFig.
3isshowninFig.
4,showingthecompletecoupledspinsystemsofthesidechains.
Forexample,allfourlysinesidechainscanbeclearlyidentified,correlatingtheNHprotondirectlywiththeterminalC~H2protons.
Similarly,thecompletespinsystemofIle-20canberecognizedimmediately.
AnalogousconnectivitiesforIle-2areconsid-erablyweakerbecauseofthebroadeningoftheNHresonance.
Nocon-nectivitiesbetweenthearomaticringprotonsofthephenylalanineresi-duesandthealiphaticprotonsareseen,althoughsmallcouplingsbetweenCaandC~protonshavebeenreportedforsuchresidues.
Incontrast,theisolatedC2HandC4Hprotonsofthehistidineresidueshowintensecor-26M.
Zasloff,Proc.
Natl.
Acad.
Sci.
U.
S.
A.
84,5449(1987).
162ADVANCEDTECHNIQUES[8]relationstoeachotherandC4HshowsacorrelationtotheC~protons.
Inourexperience,thislatterconnectivityisusuallynotobservableinmole-culessignificantlylargerthanthemagaininpeptide.
Notealsothatthecross-peakfortransferfromC,H2(F1)toC4H(FDismuchstrongerthanthecross-peakcorrespondingtotransferintheoppositedirection.
Thisiscausedbytooshortadelaytimebetweenscans:bywaitinginsufficientlylongtheslowlyrelaxingC4HprotonshavelessmagnetizationavailablefortransfertotheC,H2protonsthanviceversa.
Thistypeofasymmetryartifactcausedbytoo-shortdelaytimescanbeseeninmanytypesof2Dexperiments.
Somedistortionofthe2DspectrumofFig.
3intheregionoftheresidualwaterisseen.
ThisresidualwateralwaystendstobeamuchlargerprobleminHOHAHAexperimentsincomparisonwith,forexam-ple,NOESYorCOSYmethods.
Thereasonliesintherelativelylongtrimpulsesused.
Watermagnetizationthatisrelativelyfarremovedfromthereceivercoilandispresentinaverypoorhomogeneousregionofthemagneticfieldisexcitedbytheselongpulses,givingasignificantresponseinthe2Dspectrum.
Asdiscussedinthenextsection,analternativemethodthatdoesnotuse~Hpresaturationwillautomaticallyavoidexcita-tionofthisspuriousH20signal.
TracingConnectivitiesalongtheAliphaticSideChainsForproteinslargerthan60or70aminoacids,theT2(Tlo)valuesoftheamideprotonsoftenbecomeextremelyshort(<20msec)andthelongmixingperiodsneededforconnectingtheamideresonanceswithallside-chainresonancesnecessarilyleadtoverylowsignaltonoiseinthefinalspectrum.
However,connectivitiestotheCaandC~protonsoftenarevisible.
InthiscaseitcanbeveryusefultorecordasecondHOHAHAspectruminD20solution.
Typically,magnetizationpropagatesveryrap-idlyalongthealiphaticsidechains,andamixingtimeof35-50msecisoftensufficienttoobserveallpossibleconnectivities.
Asaresult,thespectrumbecomesverycrowded;andtodistinguishdirectfromindirectconnectivities,itmaybenecessarytorecordHOHAHAspectraforatleasttwomixingtimesortocomparethespectrumwithanabsorption-modeCOSYspectrum.
TheT2valuesofthealiphaticresonancesoftenaresubstantiallylongerthanthoseoftheamideprotonsandexcellentspectracanbeobtainedforquitelargeproteins.
Figure5showsthearomaticandaliphaticregionsofstaphylococcalnuclease,complexedwithpdTpandcalcium(18kDa).
27D.
Marion,M.
Zasloff,andA.
Bax,FEBS.
Lett.
227,21(1988).
[8]HOMONUCLEARHARTMANN--HAHNEXPERIMENTS163o.
itoI01+-8~.
ooPOt.
.
~"=:qOo:*ooOo~lO~e,o°o.
oi.
.
o~.
'0,'~_0,°+~.
~.
oi°~'.
~,.
~,,',o'o,o,o.
%o,3,-~,.
0b~.
,~~o+.
°o*+"'+goo'*'"0'U,e'Co,~p.
~,e0-''i0o0e,o^O~-0c~0O.
~'C~°".
Io;.
:0'v'l0e"c~b*.
.
.
.
°I'O~oo0,'I,.
%.
_~X.
,,3~__olo,~,,f,,Ik-9-~:~v'.
'.
'.
e~.
.
~.
0"~@o0-~/-,O~.
%0o,,.
,~oeOe~*0I~0le0O,T22oOgIIT22+--+005437'I0PPMFIG.
5,Two-dimensionalHOHAHAspectrumofthealiphaticregionofastaphylococcalnuclease/pdTp/Ca2÷complex(18kDa),1.
5mMinDzO,100mMNaCI,pD7.
4.
Thespec-trumwasrecordedwithamixingtimeof35msecandthemeasuringtimewas6hr.
Durationofthetrimpulseswas1.
5msecandthe90°pulsewidthwas28/~sec.
Labeledcross-peaksfollowtheassignmentsofTorchiaetal.
28Someoftheassignments,basedonisotopiclabelingstudies,28areindi-catedinthefigure.
Thephasedistortionobservedfortheintensereso-nanceoftheresidualHDOresonanceislargelyduetothefinitedurationofthetrimpulses.
Asmentionedearlier,coaddingdatarecordedwithtwodifferentdurationsofthetrimpulsescanremovethistypeofartifact.
Therelativeintensitiesofresonancesfromnonmobilemethylenereso-nancescanbecomeverylowifthestrongestresolutionenhancementisz8D.
A.
Torchia,S.
W.
Sparks,andA.
Bax,Biochemistry27,5135(1988).
164ADVANCEDTECHNIQUES[8]used.
Forthisreasonitmaybeusefultoprocessthedatatwice,usingdifferentline-broadeningfunctions.
Thesame,ofcourse,isalsotrueforprocessingthedatafromNOESYandCOSYspectra.
RecordingHOHAHASpectrawithoutPresaturationAsmentionedabove,presaturationofH20whenrecordingHOHAHAspectracanleadtoseriousproblemscausedbywaterfromoutsidethereceivercoil.
Moreover,presaturationobliteratesabandofCaprotonsresonatingclosetotheH20resonance.
Inaddition,whenworkingatornearphysiologicalpH,theamideexchangeratesformanyoftheresiduesaresuchthatH~Opresaturationalsosaturatestheamideresonances,whichinturnmaypartiallysaturateotherprotonsthathaveastrongNOEinteractionwiththeseamideprotons.
Forthesereasons,itmaybebettertorecordtheHOHAHAspectrumwithoutpresaturation,byusingoneofthenonexcitationwatersuppressionschemes.
Onesuchscheme29hasbeendemonstratedtofunctionwellfortheHOHAHAmethod.
OnourBrukerAM600spectrometerweprefertouseaslightvariationofthismethodwhichhasamorefavorableexcitationprofile.
ItspulsesequenceissketchedinFig.
6.
Essentially,thisisthesamesequenceastheoneofFig.
le,withthefollowingminormodifications:Afterthemixingse-quence,thespin-lockedmagnetizationisstoredalongthezaxisbymeansofthe90~flip-backpulse.
Subsequently,thezmagnetizationis"read"byaconventional1-1"jump-and-return''3°sequence.
Onehastopreventthewaterresonancefromgettinginverted,whichwouldleadtosevereradiationdampingproblems.
Therefore,thefirstexcitationpulseiscycledonlyinatwo-step(nottheregularfour-step)fashion.
Below,theproce-duretypicallyfollowedduringthesetupofthisexperimentisdescribedbriefly.
First,the90°pulseisdetermined,simplybytakingone-halfthepulsewidthofa180°pulseorone-fourththewidthofa360°pulse.
Next,theschemeofFig.
6isexecutedwiththepulsewidthsofthefinal1-1sequencesettozero.
Thewidthoftheflip-backpulseisthenfinelyad-justedtominimizetheamountofH20signal.
The1-1sequenceisthenreinstatedandthereceivergainisadjustedtoavoidoverload.
Typicallyat500or600MHz,a90°pulsewidthof30or25/zsecwillbeoptimalforcoverageofa10-ppmspectralwidth.
Morepowerwillresultintoomuchsampleheating,causinglockproblemsandtlnoise.
Also,tominimizetheeffectofunavoidablerfheating,thesampleshouldbe29A.
Bax,V.
Sklen~.
LA.
M.
Gronenborn,andG.
M.
Clore,J.
Am.
Chem.
Soc.
109,6511(1987).
30p.
PlateauandM.
Gurron,J.
Am.
Chem.
Soc.
104,7310(1982).
[8]HOMONUCLEARHARTMANN--HAHNEXPERIMENTS165FIG.
6.
PulseschemeoftheHOHAHAexperimentforrecordingspectrainH20solutionwithouttheuseofpresaturation.
Thisschemehasadditionalpulsesattheendofthemixingperiod(comparetoFig.
le).
The90,pulseflipsthespin-lockedmagnetizationbacktothezaxis.
Anadditionalsaturationpulse(notshown)maybeappliedattheendofdataacquisi-tion.
29PhasecyclingusedonourBrukerAM-600spectrometerisasfollows:th=-45°,135°;qJ=-x,x;s=x,x,y,y,-x,-x,-y,-y;Acq=x,x,y,y,-x,-x,-y,-y.
Onsuccessivet~incrementsthephase4~isincremented(fromoddtoevennumberedspectra)ordecrementedby90°(fromeventoodd).
Thisprocedurepreventsthewaterresonancefromgettingin-vertedandstillpermitsthesuppressionofaxialpeaksandtherecordingof2Dquadraturespectra.
Similarprocedurescanbeusedforrecordingthedatainthehypercomplexformat.
29broughttoasteadystatebystartingthe2Dexperimentforabout5min,immediatelyfollowedbyarestartoftherealexperiment.
Theproceduredescribedaboveisdemonstratedforasampleof4.
5mMheneggwhitelysozyme(14kDa),pH4.
2,36°,80mMNaCl.
ThefingerprintregionoftheHOHAHAspectrum,obtainedwitha36-msecmixingtimeisshowninFig.
7.
Completeassignmentsforthisprotein,basedonverycarefuldouble-quantum-filteredCOSYandRELAYexper-iments,weregivenrecentlybyRedfieldandDobson.
31Comparisonoftheirdouble-quantum-filteredCOSYspectrumwiththespectrumofFig.
7indicatesthattheresolutionsinthefingerprintregionsofthetwospec-traarequitesimilar,despitethe20%higherfieldstrengthusedfortheHOHAHAexperiment.
However,thelowestcontourlevelintheHOHAHAspectrumisfarabovethethermalnoiselevel,somuchstrongerresolutionenhancementandhigherdigitalresolutionprobablycouldbeusedtofurtherenhancespectralresolution.
Baselinecorrectionofthefinal2Dspectrumwasnotusedbutwouldenhancetheappear-anceofthespectrumsignificantly,particularlyinthelowerright-handcornerofthespectrumclosesttotheH20resonance.
Becauseoftherelativelyshortmixingperiodused(36msec),theamountofrelayobservedinthespectrumofFig.
7isrelativelylimited.
However,NHcross-peakstoboththeC~HandtheC~Hresonancesarepresentformostresidues.
ForVal-2,theentirespinsystemisclearlyvisible.
TheamountofrelayissignificantlylargerthanobservedforotherValresidues,perhapsbecausethisaminoacidattheN-terminusmayhaveincreasedmobilityofthesidechain.
NH-C~H3connectivitiesfor31C.
RedfieldandC.
M.
Dobson,Biochemistry27,122(1988).
166ADVANCEDTECHNIQUES[8]0'OV20~1oA12~I^9~Al10~-II~.
~vi)OA32~~,A42~'oA95A100AllOA82~.
Qo(~"Q@aw~',e0~',°g8oou@~voV2o0~0~,"0@00000o@oo0h"0008oo°~O"oIoO0.
.
.
,00~0A3110eo0000-col-sssA~O7v--eCj~ov~)0e"oo0.
;1o,o,o"o".
0ioo-~vOo0{}oo"o~0';'Oo_o°°.
00o0oO,'F2''8II'7PPMFIo.
7.
Two-dimensionalHOHAHAspectrumofheneggwhitelysozyme,4.
5mM,pH4.
2,75mMNaCI,35°.
ThespectrumwasrecordedwiththeschemeofFig.
6,usingamixingtimeof35msec,24-/zsec90°pulsewidth,800x2048datamatrix,acquisitiontimesof40and102msecintzandt2dimension,respectively.
Sixteenscanswererecordedperttvalueandthetotalmeasuringtimewas7hr.
AssignmentsaretakenfromRedfieldandDobson.
3~Nobaselinecorrectionprocedureofthefrequencydomainspectrawasusedandthelowest[8]HOMONUCLEARHARTMANN-HAHNEXPERIMENTS167thealanineresiduesarelabeledinFig.
1.
Atthecontourlevelshown,theserelayconnectivitiesareabsentforAla-9,Ala-31,andAla-107.
Atlowercontourlevelstheseconnectivitiesarealsoobserved,butitisnotclearwhytherelayintensitieswouldbeweakerfortheseresidues,con-sideringthatthecouplingbetweenCaandCt3protonsisalways7Hz.
Threeunidentifiedcross-peaks,notpresentintheCOSYspectrumofRef.
30(near9.
1/4.
4ppm),aremarked".
"DiscussionAppropriatehardwareisrequiredfortherecordingofhigh-qualityHOHAHAspectra.
First,anrfamplifierat1Hfrequenciesisneededthatcanproducethepowerneededfora25-to35-/~sec90°pulse(typically2-5W)andthatcanproducethisamountofrfpowerwithoutanydroopforatleast50msec.
Attenuatingthehigh"observepulsepower"isusuallynotanadequatesolutionandmayresultinburnedattenuatorsorinacollapseofthepowersupplyoftheamplifier.
Alinearornear-linearamplifierasisoftenusedfor1HdecouplingisbettersuitedfortheHOHAHAirradia-tion.
Tokeepthecomplexityofthe2Dexperimenttoaminimum,weusethesamepowerforpulses,trimpulses,andmixingsequences.
AslightlossinsensitivityandasmalllinearphaseerrorintheF~dimensionresultingfromrfoffseteffectsatthesemediumlevelsofrfpowercausenomajorproblemsinpractice.
Ifmoreeffectivemixingsequencesaredevel-opedinthefuture,itmaybecomenecessarytoswitchbetweenhigh-powerpulsesandlow-powermixingfortheirapplication,ashasbeenproposedfortheROESYexperiment,a2Atpresent,thereappearsnoneedforsuchpowerswitching.
Ifonewantstousethe~Hdecoupleramplifierforgeneratingrfpulses,thedecouplerrfmustbephasecoherentwiththereceiver.
Althoughinprinciplethislatterrequirementcanbeavoidedbyusinganinterestingz-filteredversionoftheexperiment,33inpracticethisapproachcausesextratlnoiseandwithitwehavebeenunabletogeneratespectraofaqualitysimilartothatobtainedwiththesequencedescribedinthischapter.
32C.
GriesingerandR.
R.
Ernst,J.
Magn,Reson.
75,261(1987).
33M.
Rance,J.
Magn.
Reson.
74,557(1987).
contourlevelisfarabovethethermalnoise,butclosetothebaselinedistortionscausedbyincompletewatersuppressionandbyfrequency-dependentphasecorrectionusedintheF~dimension.
Threeofthe12NH-C~H3relayconnectivitiesforAlaresidues(A31,A9,A107)arenotobservedatthiscontourlevel,despiteintenseNH-C~Hcross-peaks.
Resonancesmarked""werenotobservedintheCOSYspectrum.
168ADVANCEDTECHNIQUES[8]Ofthevariousmixingschemesdiscussedhere,theMLEV17andWALTZ17schemesappeartobemostsuitableforproteinstudies.
Alloftheearlierschemes(Fig.
la-c)givestrongadditionalNOEcross-peakswhichcanbeveryconfusingandwhichalsocandecreasethesensitivityoftheexperimentsignificantlyincaseswhereNOEandHOHAHAef-fectscompete.
ComparisonoftheMLEV17andWALTZ17schemesre-vealsafewdifferences.
Fromapracticalpointofview,theMLEV17mixingschemeappearstobelesssensitivetoimperfectionsinthephase-shiftinghardware.
However,thebandwidththatcanbecoveredwithMLEV17foragivenamountofrfpowerisabout25%smaller.
WALTZ17mixinghastheintrinsicdisadvantagethattheeffectivesizeofthecou-plingduringmixing(andthusthetransferrate)isreducedmorethanitisfortheMLEVsequence.
Foroffsetsusedinpractice(smallerthanv~f/2),thiseffectissmall.
ThemaximalbandwidthcoveredbytheWALTZ17sequenceisabout-+0.
4v~fandforMLEV17,about-+0.
33v~f.
Formostdiamagneticproteinsthisallowseffectivecross-polarizationovertheen-tirespectralrangewithonlyafewwattsofrfpower.
Overall,ifspectrom-eterhardwareissufficientlygood,theWALTZsequenceappearsprefera-bleovertheMLEVmixingscheme.
Two-dimensionalHOHAHAexperimentsareparticularlyusefulfordelineatingindividualspinsystemsinproteins.
Themethodprovideshigh-sensitivity,high-resolutionspectrathatcontainawealthofrelayedconnectivityinformation.
TherearetworeasonswhyHOHAHAspec-troscopymaybemoresensitivethanconventionalCOSY-typeexperi-ments.
First,becauseofthein-phasenatureofthetransferofmagnetiza-tion,cancellationofabsorptivecomponentswithinacross-multipletdoesnotoccur.
Second,nomagnetizationislosttothegenerationofspuriousmultiple-quantumcoherence,amajorsourceofsensitivitylossinconven-tionalRELAYexperiments.
Resolutioninthefingerprintregionofdou-ble-quantum-filteredCOSYandHOHAHAspectraisquitesimilar;al-thoughinourexperience,thesensitivityoftheHOHAHAspectraistypicallymuchhigher.
Forstudyingspectralregionsclosetothediagonal,theHOHAHAspectrumusuallyoffersthebestappearancebecauseoftherelativelylowintensityofthediagonal.
AcknowledgmentIthankLauraLernerformanyusefulcommentsduringthepreparationofthismanu-scriptandRolfTschudinfortechnicalassistanceinmodifyingspectrometerhardware.
ThenucleasesamplewaskindlyprovidedbyDennisTorchiaandStevenSparks,andthema-gainin-2samplebyMichaelZasloff.
IalsowishtoacknowledgestimulatingdiscussionswithMariusCloreandAngelaGronenborn.