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