IntroductiontoProteinNMR
Bioc530
November7,2016
LearningGoals
• HaveabetterunderstandingofNMRdatainpublications
• Determinehow/ifNMRcanbeusefulinyourproject
WritedowntheadvantagesofstudyingproteinsbyNMR.
Atomicresolutionofstructureanddynamicsinsolution• Onlywaytodetermine3Datomicresolutionstructureinsolution• Studyprotein-proteinorprotein-ligandinteractions,includingveryweakinteractions.
• Measuretimescalespecificbackboneandsidechainflexibility• Detectlowlypopulatedconformations
WhyProteinNMR?
WritedownthedisadvantagesofstudyingproteinsbyNMR.
• The protein must be in a well-defined oligomeric state• 0.5 - 1 mM is the optimum protein concentration for structural and
dynamical studies• The NMR sample should be stable over periods of time required to
collect the NMR data (days-weeks-months)• pH < 7 is preferred, as it minimizes the loss of 1H sensitivity due to
exchange with water protons.• Overwhelming majority of the proteins studied by NMR are over-
expressed in and purified from E. Coli• M9 (minimal media) with 13C-enriched glucose and 15N-enriched ammonium chloride
as sole carbon and nitrogen sources is used for 13C/15N labeling• E. Coli growth in D2O is used to introduce deuterium into non-exchangeable protein
sites. Partial deuteration is required for NMR studies of proteins > 25 kDa
WhyNotProteinNMR?
TypicalNMRspectrometersetup
“TheMagnetisalwaysON”
Magneticfieldstrength11.74Tesla(500MHzforproton)othercommonfieldstrengths600or800MHz
NuclearMagneticResonancespectroscopy
• Nucleushasaspin,whenyouhaveaspinningchargethereisaninducedmagneticdipole
• Notallnucleihavespin
SpinQuantumMechanicsTheverybasicsofNMR
Nucleiwithmagneticdipole
Nuclei Unpaired Protons
Unpaired Neutrons Net Spin, I % Natural
Abundance γ (MHz/T)
1H 1 0 1/2 99.9985 42.582H 1 1 1 0.0115 6.5412C 0 0 0 98.9313C 0 1 1/2 1.109 10.7114N 1 1 1 99.636 3.0815N 0 1 1/2 0.364 -4.3619F 1 0 1/2 100 40.0831P 1 0 1/2 100 17.235
Evennumberofbothprotonsandneutrons,I = 0Sumofprotonsandneutronsisodd,I = 1/2, 3/2, 5/2, …Odd number of both protons and neutrons, I = 1, 2, 3, …
Needtoenrichsampleswith13Cand15Nsincelownaturalabundance(moreonthislater)
Determiningthespinofnuclei
MostinterestedinnucleiofspinI =½(magneticdipole)
• I =1/2hastwopossibleenergystates,m=± 1/2
• Inthepresenceofanexternalmagneticfield,eachnucleicanalignwith(‘spinup’,lowenergy)oragainst(‘spindown’,highenergy)theexternalfield(B0)
TheverybasicsofNMR
ΔE=hν,ν fallsinradiofrequencyregionofelectromagneticspectrum;γ = gyromagnetic ratio (see previous table)ν = γB0 istheLarmor frequency(denotedω)
PopulationofstatesaccordingtoBoltzmandistribution:
IncreasespinexcessbyloweringTorincreasingexternalfieldstrengthB0
!"#$"%!'()%
= 𝑒,-./ħ1.2
Nucleiwithmagneticdipole
LowE HighE
• Larmor precession:becausenucleirotate,nuclearmagneticfieldwill‘precess’aroundtheaxisoftheexternalfieldvector(thisisanangularmomentumthing,lookupvideosonspinningbikewheelsifyouwanttovaguelyrelateittosomethingphysical)
• WecandetectsignalsintheX-YplaneApplicationofRFpulse(attheLarmor frequency)perpendiculartoexternalfieldpushesthemagnetizationintotheX-Yplane
TheverybasicsofNMR
B0
+
B0
z
y
x
zω = γB0
Transmitter/ReceivercoildetectssignalinX-Yplane
FreeInductionDecay(FID)Signaloscillatesanddecays
overtime
ω = γB0
FT
ω
Our‘peak’
Bothpeaklocationandwidth(dynamics)areimportant
Oursignalappearsatsomefrequency,dependentonthemagneticfieldstrength
Tomakelifeeasier,weworkwith‘chemicalshift’insteadoffrequency(mostly)d =(n - nREF)x106 /nREF inunitsofppm(partspermillion;fieldindependent)
SpinQuantumMechanicsChemicalShift
B0
z
y
x
ReceivercoildetectssignalinX-Yplane FreeInductionDecay(FID)
ω = γB0
FT
ω
Our‘peak’
widthdependsonT2relaxation– largerproteinshavebroaderpeaks(>30ishkDa andtricksbecomenecessary)
ApplicationofRFpulsesofspecifiedlengthsandfrequenciescanmakecertainnucleidetectable
Wecanselectivelyexcitenucleiofinterest.
1DNMRspectra
Signalsfromall1Hofsomefoldedprotein
H-N H-C
Water
ApplicationofRFpulsesofspecifiedlengthsandfrequenciescanmakecertainnucleidetectable
Wecanselectivelyexcitenucleiofinterest.
1DNMRspectra
Signalsfromall1Hofanunfoldedprotein
Significantlylessdispersioninamideregionlossofuniquechemical/structuralenvironments
H-N H-C
Water
Chemicalshiftisexquisitelydependentonnuclei’schemical/electronicenvironmentsNucleiaresensitivetonearbynucleiScalarcoupling(J)isathrough-bondeffect:spinofonenucleusperturbsspinsofinterveningelectrons…..CausessplittingoftheNMRsignal;containoodlesofinfo
Chemicalshiftandscalarcouplings
3Jcouplingscontaintorsionangleinformation(e.g.,HN-Hα forbackbone,C’-Cγ orN-Cγ forsidechains,manyothercombinationspossible&measurable)
StructuralInformationfromJ-couplings
3JCγN
3JCγCO
Predicted 3J values
χ1 = 180o χ1 = +60oχ1 = -60o
Measured by NMR Karplus curves
MultidimensionalNMR
1DNMRgivessignalsofjustonenuclei(e.g.1H,13C,or15N)Muchmoreinformationwhenweadddimensions.Weusethethrough-bondJcouplingstopassaroundthemagnetization
Mostfrequentlyused2DNMRspectraistheHSQC(heteronuclearsinglequantumcoherence)MagnetizationistransferredfromtheHtotheattached15NnucleiviatheJ-coupling
StackedPlot
1H
15N
intensity
2DSpectra
ContourPlot
NMRorNotNMR
• Toseeifyourunlabeledproteinisfolded• Todeterminethestructureofan8kDa proteinwithflexibleloops
• Todeterminethestructureofa30kDa wellfoldedprotein
• Tostudyintrinsicallydisorderedproteins• Todeterminewherealigandbindstoaprotein• Tostudylargeproteincomplexes• Tostudymembranebindingproteins• Tostudymultipleconformationssampledbyaprotein
References
Goodoldschool,shortintrovideoonnuclearspin(otherepisodesaregood,too)https://www.youtube.com/watch?v=jUKdVBpCLHM
UCDavisNMRwiki(sourceofspingraphics)http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Magnetic_Resonance_Spectroscopies/Nuclear_Magnetic_Resonance/Nuclear_Magnetic_Resonance_II
DukeintrotoNMRhttp://www.cs.duke.edu/brd/Teaching/Bio/asmb/current/2papers/Intro-reviews/flemming.pdf
ExcellentpracticalguideforNMRexperiments(pulseprograms&howtheywork)http://www.protein-nmr.org.uk/
NMRAssignments– AsimpleexampleassigningasmallIntrinsicallyDisorderedPeptide
Backboneamides
Asn/GlnsidechainNH2TrpsidechainNH(foldedin15N)
15N-HSQC
1) Protein sample preparation• Overwhelming majority of the proteins studied by NMR
are over-expressed in and purified from E. Coli
• M9 (minimal media) with 13C-enriched glucose and 15N-enriched ammonium chloride as sole carbon and nitrogen sources is used for 13C/15N labeling
• E. Coli growth in D2O is used to introduce deuterium into non-exchangeable protein sites. Partial deuteration is useful for NMR studies of proteins > 25 kDa
• Insect cell medium and in-vitro translation systems enriched with stable isotopes are available; but still prohibitively expensive
2) Optimization of sample conditions
• Buffers with non-negligible temperature dependence of pH (e.g. Tris) should be avoided.
• pH < 7 is preferred, as it minimizes the loss of 1H sensitivity due to exchange with water protons.
• The protein must be in a well-defined oligomeric state• 0.5 - 1 mM is the optimum protein concentration for
structural and dynamical studies• The NMR sample should be stable over periods of time
required to collect the NMR data• days > binding studies• weeks > assignments or dynamics• months > all atom assignments / full dynamics characterization
Characteristic amino acid proton and carbon chemical shifts
Backboneamides
Asn/GlnsidechainNH2TrpsidechainNH(foldedin15N)
15N-HSQC
NMRAssignments– AsimpleexampleassigningasmallIntrinsicallyDisorderedPeptide
Backbonetripleresonanceexperiments(need1H,13C,15Nsample)
i andi-1 peaks i-1 peaks
13C(Cα,Cβ,C’)
3Dspectraforbackboneassignments
15NPlane‘2Dstrip’
BackboneAssignments– Step1:Pickthepeaks
HN(CO)CA HNCA
BackboneAssignments– Usuallylookat2Dstripstakenfrom3Dexperiment
Cαi
Cαi-1
pk#1 pk#2 pk#3
13C
BackboneAssignments
HN(CO)CA HNCA (probably)C-termD134pk#4 pk#5
13C
BackboneAssignments
HN(CO)CA HNCA (probably)C-termD134
LookforstripwithCαi peakatthisshift
Havetostartsomewhere...
pk#4 pk#5
13C
BackboneAssignments
HN(CO)CA HNCA
Closebuti-1noti peak
pk#6 pk#7 pk#8
13C
BackboneAssignments
HN(CO)CA HNCA
Winner
D133pk#1 pk#2 pk#3
13C
BackboneAssignments
pk#6pk#1 D134
D133?
CanconfirmwithHNCACB
Cαi
Cαi-1
Cβi-1
Cβi13C
BackboneAssignments
D134D133T132T131V130
ProX
Chainstopshere
BackboneAssignments
Alanine118or125?
Lookfori-1peaks
Lookforipeaks
Alanines havedistinctiveCβ shifts
PeakisA125ifthenextstriplookslikeaThr
PeakisA118ifthepreviousstriplookslikeaSer
SodoThr&Ser
Threonine
BackboneAssignments
Alanine118or125?125 T126F124
Keepfindingtheconnections
Repeatforremainingsections...
BackboneAssignments:HN,N,Ca,Cb,C’
BackboneamidesallassignedAlsoknow:Ca&Cbshifts
TrivialtoaddtheC’shifts:HNCO
13C
170
175
102 103 104 105 107 108 109 110
Sidechainassignments
13C-HSQC
Cα
Cβ (Ser & Thr)
CH3
β/γ CH2
Ca&CbareknownDon’tknowHa,Hb,...
Sidechainassignments15N-TOCSY(flattened)
Amidesondiagonal
Sidechainprotons
Hα
Hβ/γ
Methyls
1H
HN
15N
HNCACB 15N-TOCSY
13C-CHSQC
T102
Ca
Ha
Sidechainassignments
HNCACB 15N-TOCSYT102
Cb
Hb
Sidechainassignments
13C-CHSQC
Sidechainassignments
13C-CHSQCmethylregion Hg
**Don’texplicitlyhaveCgbutHgshiftisenoughtoassignforthispeptide
T102
Sidechainassignments
13C-CHSQCmethylregionA118 A125
**Cβ’swouldbesufficienttoassignthealaninesforthispeptide
Sidechainassignments:Ha,Ca,Hb,Cb,Hg,Hd...Cg,Cdinferred
Forthispeptide:CanunambiguouslyassignprettymucheverythingexceptsomeCH2γ groups&thearomatics(notshown)
MoreExperimentsrequiredforlargersystems:
13C-NOESYHCCH-TOCSY&HCCH-COSYCmCgCbCaHN....Andothertricksasnecessary