11

Paramagnetic Paramagnetic NMR: a NMR: a versatile versatile tool tool in in

structural biologystructural biology

Claudio LuchinatClaudio Luchinat

CERMCERMUniversity of FlorenceUniversity of Florence

800800700b700b

600600

850ss850ss

700700

400400

500500

BioBio--labslabs

LibraryLibrary

700ss700ss

900900

GENEXPRESS, CRYST, CISMGENEXPRESS, CRYST, CISMDepartment Department of of ChemistryChemistry((officesoffices, , biobio--labslabs, , relaxometerrelaxometer,, instrumentsinstruments..)..)

WorkshopWorkshop

ConferenceConference roomroom

600b600b

The Magnetic Resonance Center in FlorenceThe Magnetic Resonance Center in Florence

Computer room Computer room

Electron/Electron/nuclear relaxationnuclear relaxation (Relaxometry)(Relaxometry)Drug discoveryDrug discovery

Structural proteomicsStructural proteomicsMetabolomicsMetabolomics

Protein structure determinationProtein structure determinationMethodological advancementsMethodological advancements in NMRin NMRSolidSolid state NMRstate NMRBioinformatics andBioinformatics and computational biologycomputational biology

22

EUEU--NMRNMR –– EuropeanEuropean Network ofNetwork of Research Infrastructures for Research Infrastructures for providingproviding Access andAccess and Technological AdvancementsTechnological Advancements inin biobio--NMRNMR

FlorenceFlorence

FrankfurtFrankfurtUtrechtUtrechtBirminghamBirmingham

LyonLyon--GrenobleGrenoble

ABI-AAD, EABOUKAIS, AADACHI, HAHN, HCAIME, SALCANTARA, RANDERSEN, RAANSARI, AAAOKI, DARCON, DBABASHKINA, MGBAHO, NBAKHMUTOV, VIBALAYSSAC, SBALICAS, LBANCI, LBARONE, VBARRON, ARBAUMGARTEN, MBERG, DJBERMEL, WBERNINI, ABERRY, REBERTHIER, CBERTINI, IBERTONCINI, CWBEZSONOVA, IBIANCHINI, CBILL, EBINNEMANS, KBINOLFI, ABOBROFF, JBOHLMANN, WBONDON, ABONG, PHBOREL, ABRACCI, LBRINGMANN, GBROWN, SEBRUCHER, EBRYANT, RGBRYLIAKOV, KPBUBACCO, LCAI, SCAMPOS-OLIVAS, RCANNY, J

ParamagneticParamagnetic & NMR in ISI in 2006& NMR in ISI in 2006--2007 2007 (at(at leastleast 2 2 publicationspublications))

CANTERS, GWCAPOZZI, FCARAVAN, PCHEN, YCHRISTENSEN, HEMCHRISTLIEB, MCIMINO, PCIOFI-BAFFONI, SCLARK, WGCLEARFIELD, ACLORE, GMCOLNAGO, LACOSTA-COQUELARD, CCOURCOT, DCRESCENZI, ODA COSTA, GDELVILLE, ADENG, YMDENNISON, CDESREUX, JFDIAKOVA, GDIXON, NEDONOSO, JPDORN, HCDOTSCH, VDUBAN, EADUBOIS, MDUCHAMP, JCEDWARDS, CLELST, LVEMSLEY, LENDERS, MENDO, KENGELKE, FENKELMANN, VEVANICS, FFABER, CFABER, JHFAUGERE, AMFAWAL, ZFEDUSHKIN, ILFELLI, ICFERNANDEZ, COFIEDLER, JFLORKE, UFORMAN-KAY, JD

FRANCESCONI, LCFRANCHI, PFRIES, PHFUJII, HFUJIMOTO, TFUKIN, GKFUNASAKI, NFURUTANI, YGAGGELLI, EGALEZOWSKA, JGARCIA, GGARCIA, LGARCIA-ESPANA, EGERALDES, CFGCGIAMBASTIANI, GGIANOLIO, EGIPPIUS, AAGIRAUDET, JGRAZIANI, OGRECH, EGRIESINGER, CGRIFFIN, RGGRIFFITHS, PCGRONENBORN, AMGRONENBORN, BGUERIN, KHAGA, YHAHN, FEHAMON, JRHAMWI, AHANSEN, DFHASS, MASHAYASHI, SHEENAN, RKHEIDEMANN, DHELM, LHIRATA, HHOMMA, YHORSTMANN, MHORVATIC, MHU, KNHUANG, WHUBER, THUMMERT, MICHIOKA, MIFTIKHAR, K

IKEZAKI, AISHII, YIWAHARA, JJACQUES, VJENSEN, MRJEONG, SYJERSCHOW, AJIMENEZ, HRJOHN, MJONES, CEJOSHI, JDKACZMAREK, SMKAIM, WKALBITZER, HRKAMBE, SKAPADIA, MAKARIM, CBKERVERN, GKHVOINOVA, NMKIMPE, KKITEVSKI, JLKLEIN, SIKOBAYASHI, AKOBAYASHI, HKOBAYASHI, YKOHLER, FHKOLODZIEJ, BKORB, JPKOVACS, ZKOZERSKI, LKOZLOWSKI, HKRUK, DKUHN, LTKUPPUSAMY, PLA MAR, GNLATOS-GRAZYNSKI, LLAURENT, SLAVELA, PLED, JJLEE, WTLENIEC, GLEROUX, FLEZNOFF, DBLI, HHLIANG, BYLIM, AR

LIU, GCLONGO, DLUCARINI, MLUCHINAT, CMA, LHMACHIDA, KMAGON, CJMANGIAPIA, GMARKLEY, JLMARSH, JAMARTIN-NETO, LMASIN, FMATSUMORI, NMAZZANTI, MMEIER, RMELI, AMERBACH, AEMEZZINA, EMICHOT, LJMIZUSHIMA, TMLYNARZ, PMOLTENI, EMONTGOMERY, LKMOROOKA, AMORTILLARO, AMOSKVIN, ASMUGNIER, YMULLER, RNMURATA, MNAAL, ZNAG, KNAKAMURA, MNAKANISHI, WNICCOLAI, NNISHIHARA, HNOESKE, JNONAT, AOBERHAUSER, WOHGO, YOLDFIELD, EONUKI, YORTIZ, GFOTTING, GOVERHAND, MPACHOLSKA-DUDZIAK, EPADUANO, L

PANICH, AMPARAC-VOGT, TNPARIGI, GPARK, AYPARKER, DPATEL, GPPATEL, MMPATEL, NBPATEL, SDPAUL, APAVONE, MPAZDERSKI, LPECAUT, JPEREZ, JPERROTIN, PPETERS, JAPETRYAKOV, SPICCIOLI, MPIERATTELLI, RPINTACUDA, GPOLENOVA, TPOPPL, APORCIATTI, EPORION, PPRESTEGARD, JHPRISCHI, FPROSSER, RSRACHLEWICZ, KRAFTERY, DRAJADURAI, CRANIN, PRASIA, RMRATH, SPREICHERT, YREYES-GARCIA, EARHEE, JYRIOS, IGRIVERA, MRODRIGUEZ, JCRUHLMANN, LRUSSO, PLSAFARI, NSAFIN, DASAKAI, HSAMOUILOV, ASANTANA, MD

SARKAR, BSATO, KSCHILF, WSCHMITZ, CSCHUMANN, HSCHURKO, RWSCHWALBE, HSCHWEDHELM, KFSGHERI, LSHAIBAT, MASHAMES, AISHAPIRO, PJSHERRY, ADSHIMIZU, TSHIOKAWA, YSHPEIZER, BGSINGH, PSINNEMA, PJSITKOWSKI, JSMIRNOVA, TISMITH, KMSOKOLOV, FDSORACE, LSORIANO, CSPIGA, OSPILLER, MSRIVASTAVA, ASRIVASTAVA, SSUN, YPSZLYK, ETAKAGI, HTAKAHASHI, MTALSI, EPTAMM, LKTANG, CTENG, CLTHOMAS, DDTHOUVENOT, RTIAN, FTIERNEY, DLTILSET, MTIRADO, JLTIRCSO, GTOKUNAGA, YTOTI, ATOUPET, L

TOUSEK, JTRONTO, JTURANO, PTYPEK, JUBBINK, MUEDA, YVALENSIN, DVALENSIN, GVALIM, JBVAN ELDIK, RVEGA-ROCHA, SVEGLIA, GVENDITTI, VVENOT, AVERDEJO, BWADA, SWALKER, FAWALSTEDT, REWALTER, MDWANG, YMWARREN, JEWASYLISHEN, REWICKRAMASINGHE, NPWILKS, AWOHNERT, JWOODS, MWORRALL, JARWU, GQXIE, YYAMAMOTO, YYOSHIDA, TYOSHIMURA, KZABIROV, NGZAHL, AZARGARIAN, DZHANG, HMZHAO, PYZOPPELLARO, GZORKO, AZWECKSTETTER, MZWEIER, JL

33

NMR and EPR NMR and EPR signalssignals

Experimental Experimental NMR NMR signalsignal

Experimental Experimental EPR EPR signalsignal

00 BBgh NNN γµν h==

0Bgh Beµν =

NγhNg11HH 5.595.59 2.81 2.81 ××1010−−26 26 JTJT−−11

22HH 0.860.86 0.430.4333HH 5.965.96 2.992.991313CC 1.401.40 0.710.711414NN 0.400.40 0.200.201515N N −−0.57 0.57 −−0.280.283131PP 2.262.26 1.141.14

=Beg µ 1.86 1.86 ××1010−−23 23 JTJT−−11

400 400 MHz MHz 11H H Larmor frequencyLarmor frequency==

263 263 GHz GHz of electron of electron Larmor frequencyLarmor frequency

EPRe− (592 GHz)

1H (900 MHz)NMR

The The hyperfine couplinghyperfine coupling

α

β

EPR

νa

νβ

Electron Zeeman

Nuclear Zeeman

Hyperfine| βS,αI>

| βS, βI>

| αS,βI>| αS,αI>

A/

A/

h

h

The coupling constant between nucleiI1…I2 is called J

The coupling constant between nucleus and e-

I1…e- is called A

⇒the interaction is expressed by AI·S

44

Nuclear Nuclear and electronand electron relaxation timesrelaxation times

Carboxyl carbon Carboxyl carbon of of glycinesglycines:: 44 s44 s

Methyl protons Methyl protons of of ethylbenzene ethylbenzene (in CDCl(in CDCl33):): 10 s10 s

Water Water protonsprotons:: 3 s3 s

Ethanol protonsEthanol protons:: 1.7 s1.7 s

CHCH22 protonsprotons inin trioleintriolein:: 0.7 s0.7 s

Amide protons Amide protons in in proteinsproteins of 10,000 Da: of 10,000 Da: ∼∼0.5 s0.5 s

Methylene protons Methylene protons in in proteins proteins of 10,000 Da: of 10,000 Da: ∼∼0.3 s0.3 s

Electron Electron relaxation relaxation time of a time of a radicalradical: 10: 10--77 s (TEMPOL)s (TEMPOL)N

O

•

Electron Electron relaxation timesrelaxation times of metal of metal ionsions: 10: 10--88 -- 1010--1212 ss MMnn++

EPRe− (592 GHz)

1H (900 MHz)

MI

NMR

The The hyperfine shifthyperfine shift

α

β

EPR

νa

νβ

Electron Zeeman

Nuclear Zeeman

Hyperfine| βS,αI>

| βS, βI>

| αS,βI>| αS,αI>

A/

A/

δ

KTSSgA

I

Be

γµδ3

)1( +=h

h

h

The coupling constant between nucleiI1…I2 is called J

The coupling constant between nucleus and e-

I1…e- is called A⇒the interaction is expressed by AI·S

Since A is anisotropicthe interaction is expressed by I·A·S

55

The The hyperfinehyperfine shift (CS+PCS)shift (CS+PCS)CS = contact shift

PCS = pseudocontact shiftZn

Contact Contact shiftsshifts

kTBSSgS eBz 3

)1( 0+−>=< µSI ⋅= AH

Kurland and McGarvey (1970) predict that <Si> and hence contact shift may be orientation dependent due to spin-orbit coupling

H.M. McConnell, D.B. Chesnut, J.Chem.Phys. 1958, 28, 107-117

R.J. Kurland, B.R. McGarvey, J.Magn.Reson. 1970, 2, 286-301

I. Bertini, C. Luchinat, G. Parigi.I. Bertini, C. Luchinat, G. Parigi. EurEur. J. . J. InorgInorg. Chem.. Chem. 24732473--2480, 2000. 2480, 2000.

66

PPseudocontact shiftsseudocontact shifts

The Curie spin – nuclear spin interaction energy averages zero upon rotation

Curie interaction

SSzz

rτ SSzzrτ

SSzz

rτ SSzzrτ

( ) 0sin)1cos3(2

0

21 ≠−∝ ∫

πγγγγµ dE

Isotropic Sz

Anisotropic Sz

Consequences:1) The Curie spin – nuclear interaction energy does not average zero upon

rotation

( ) ⎥⎦

⎤⎢⎣

⎡Ω−+−⎟⎟

⎠

⎞⎜⎜⎝

⎛ +−= 2cossin

23)1cos3(

21

121 22

3pcs θχχθ

χχχ

πδ yx

yxzr

0sin)1cos3(2

0

2 =−∝ ∫π

γγγ dE

Pseudocontact Pseudocontact shiftsshifts

( ) ( )⎥⎦⎤

⎢⎣⎡ ∆+−∆=

−

φθχθχδ 2cossin231cos3 22

3metalrh

metalax

MX

pcs

rK

θθ

ϕϕ

χχzzzz

χχyyyyχχxxxx

XXzzyy

yy

rrMM

Surfaces with constant δpcs values:Axial Totally Rhombic

0rh =∆χ axrh )3/2( χχ ∆=∆

positivenegative

B0 µ1

µ2

r

( )( )⎥⎦⎤

⎢⎣⎡ ⋅

−⋅⋅

−= 350 3

4 rrH 2121 µµrµrµ

πµ

IµI Iγh=

0

µ0Bµ ⋅

=><χ

77

Partial Partial selfself--orientationorientation

bilayer micelle bicelle

Orientation induced by restriction in spaceB0

Energy profile of the magnetic interaction energy with the external magnetic field B0 of a

paramagnetic molecule possessing magnetic susceptibility anisotropy

χχzzzz

χχxxxx

χχyyyy

BBoo

ϕϕ

θθ1515NN

11HH

( ) ( )⎥⎦⎤

⎢⎣⎡ ∆+−∆×

×−=

φθχθχ

πγγ

π

2cossin231cos3

41541)(

22

32

20

molrh

molax

AB

BA

rh

kTBHzrdc

( ) ( )⎥⎦⎤

⎢⎣⎡ +−×

×−=

φθθ

πγγ

πµ

2cossin231cos3

24)(

22

320

rhax

AB

BA

DD

rhHzrdc

External orienting media Self-orientation

Nuclear relaxationNuclear relaxation by electron spins by electron spins

zz

yyxx

SSzz

yyxx

zz

yyxx

zzBB00

Pictorial decomposition of the electron spin magnetic moment

Decomposition of the electron-nucleus dipolar interaction

1111 −−−− ++= Mrsc ττττDipole-dipole interaction

111 −−− += MrCurie τττCurie interaction

SSzz

( )⎥⎦

⎤⎢⎣

⎡+

++

+⎟⎠⎞

⎜⎝⎛= 22226

22220

1 13

171

4152

cI

c

cS

cBeIM r

SSgRτω

ττω

τµγπ

µ2262

2244220

13

)3()1(

452

CurieI

CurieBeI

rkTSSg

τωτµω

πµ

++

⎟⎠⎞

⎜⎝⎛+

( )⎥⎦

⎤⎢⎣

⎡+

++

++

⎟⎠⎞

⎜⎝⎛= 22226

22220

2 13

11341

4151

cI

c

cS

cc

BeIM r

SSgRτω

ττω

ττµγπ

µ⎟⎟⎠

⎞⎜⎜⎝

⎛+

++

⎟⎠⎞

⎜⎝⎛+ 2262

2244220

134

)3()1(

451

CurieI

CurieCurie

BeI

rkTSSg

τωττµω

πµ

Solomon Curie

88

Structure calculations withStructure calculations withparamagnetismparamagnetism--based restraintsbased restraints

Classical + Paramagnetism-based (R1, CS, PCS, RDC…) restraints

Metal ion and magnetic susceptibility tensor

The position of the metal is determinedwithout any assumption

Paramagnetic proteinParamagnetic protein

Diamagnetic proteinDiamagnetic protein

Classical restraints (NOE, dihedral angles, H-bonds…)

( )⎥⎦

⎤⎢⎣

⎡+

++

+⎟⎠⎞

⎜⎝⎛= 22226

22220

1 13

171

4152

cI

c

cS

cBeIM r

SSgRτω

ττω

τµγπ

µ

Nuclear relaxationNuclear relaxation rate rate restraintsrestraints

1111 −−−− ++= Mrsc ττττ

2262

2244220

13

)3()1(

452

rI

rBeI

rkTSSg

τωτµω

πµ

++

⎟⎠⎞

⎜⎝⎛+Solomon

Curie

Solution structure of Clostridium pasteurianum ferredoxinin the proximity of Cluster II Bertini, Bertini, DonaireDonaire, Luchinat, , Luchinat, Rosato Rosato ProteinsProteins, 1997, 1997

Without R1 restraints With R1 restraints

99

Contact shiftContact shift restraintsrestraints: : ironiron--sulfur proteinssulfur proteins

kTSSgAS

BA

N

Bcz

I

ccon

γµ

γνν∆

3)1(22

00

+⎟⎠⎞

⎜⎝⎛=><⎟⎟

⎠

⎞⎜⎜⎝

⎛−=⎟⎟

⎠

⎞⎜⎜⎝

⎛hh

HHββ of of ironiron--coordinatedcoordinated cysteinescysteines

Bertini, Bertini, CapozziCapozzi, Luchinat, Piccioli, , Luchinat, Piccioli, VilaVila,,J. J. AmAm. . ChemChem. Soc.,. Soc., 19941994

FeFe e-HH

SS CC θθδδ ∝∝ sinsin2 2 θθ

θθ = M= M--SS--CC--HH

Contact shiftContact shift restraintsrestraints:: unpairedunpaired electronelectron spinspin density density on heme nuclei on heme nuclei isis a function ofa function of axialaxial ligandligand orientationorientation

8-CH3 5-CH3 1-CH3 3-CH3

φ = 0° φ = 30° φ = 45°

M80A cyano-cytochrome c

I. I. BertiniBertini, C. Luchinat, G. , C. Luchinat, G. ParigiParigi, F.A. Walker, , F.A. Walker, JBICJBIC 19991999

( ) ( )[ ] βφθφθβδ sincossincos 22 dcba iii ++++−=

I II

IIIIV

xy

θ1

θ3

θ5θ8

13

58 β

φ

-φ

π

gxx

Methyl protons in histidine cytochromes (Low spin FeLow spin Fe(III))(III))

1010

( ) ( ) ( )⎭⎬⎫

⎩⎨⎧

−+−⎥⎦⎤

⎢⎣⎡ +−= φθχχθχχχ

πδ 2cossin

231cos3

21

121 22

3 yyxxyyxxzzi

pc

r

NOE-only

+ 280 pcs values

BanciBanci, Bertini, , Bertini, BrenBren, , CremoniniCremonini, , GrayGray, Luchinat, Turano, Luchinat, TuranoJBICJBIC 1, 117 (1996)1, 117 (1996)

Pseudocontact shiftPseudocontact shift restraintsrestraints

χkk

χxxχyy

χzzSolution structure of M80A cytochrome c-CN

Energy profile of the magnetic interaction energy with the external magnetic field B0of a paramagnetic molecule possessing

magnetic susceptibility anisotropy

SelfSelf--orientation residual dipolar orientation residual dipolar coupling restraintscoupling restraints

BanciBanci, , BertiniBertini, Huber, Luchinat, , Huber, Luchinat, RosatoRosato J. Am. Chem. Soc., J. Am. Chem. Soc., 19981998

( ) ( )⎥⎦⎤

⎢⎣⎡ ∆+−∆−= φθχθχ

πγγ

π2cos

231cos3

41541)( 22

32

20 sin

rh

kTB

Hzrdc molrh

molax

HN

NH

∆χaxpara = 2.8 ⋅ 10-32 m3

∆χrhpara = -1.1 ⋅ 10-32 m3

χχzzzz

χχxxxx

χχyyyy

BBoo

ϕϕ

θθ1515NN

11HH

1111

•• 11793793 NOEsNOEs•• 57 phi values57 phi values•• 46 46 psi psi valuesvalues•• 30 30 HbondsHbonds•• 13 1D13 1D--NOENOE (RMSD=0.69Å)(RMSD=0.69Å)Paramagnetic constraints:Paramagnetic constraints:

•• 1164 1164 pcspcs from 11 from 11 lanthanideslanthanides•• 2626 TT11 valuesvalues•• 254 254 rdcrdc from 7 from 7 lanthanideslanthanides•• 50 50 ccrccr + 5 + 5 cscs from from CeCe(III)(III) (RMSD=0.26 Å)(RMSD=0.26 Å)

Diamagnetic constraints:Diamagnetic constraints:

Calbindin: a summary for all Calbindin: a summary for all rerestraints included in straints included in

PARAMAGNETIC PARAMAGNETIC DYANADYANA/CYANA/CYANA

Bertini, Cavallaro, Cosenza, Kummerle, Luchinat, Piccioli, PoggiBertini, Cavallaro, Cosenza, Kummerle, Luchinat, Piccioli, Poggi, , J.Biomol.NMRJ.Biomol.NMR 20022002Barbieri, Bertini, Cavallaro, Lee, Luchinat, Barbieri, Bertini, Cavallaro, Lee, Luchinat, J.Am.Chem.Soc.J.Am.Chem.Soc., 2002, 2002Bertini, Donaire, Jiménez, Luchinat, Parigi, Piccioli, Poggi, Bertini, Donaire, Jiménez, Luchinat, Parigi, Piccioli, Poggi, J.Biomol.NMRJ.Biomol.NMR 20012001Bertini, Lee, Luchinat, Piccioli, Poggi, Bertini, Lee, Luchinat, Piccioli, Poggi, ChemBioChemChemBioChem, 2001, 2001Bertini, Janik, Lee, Luchinat, Rosato, Bertini, Janik, Lee, Luchinat, Rosato, J.Am.Chem.Soc.J.Am.Chem.Soc., 2001, 2001Bertini, Janik, Liu, Luchinat, Rosato, Bertini, Janik, Liu, Luchinat, Rosato, J.Magn.Reson.J.Magn.Reson., 2001, 2001AllAllegrozzi, Bertini, Janik, Lee, Liu, Luchinat, egrozzi, Bertini, Janik, Lee, Liu, Luchinat, J.Am.Chem.Soc.J.Am.Chem.Soc., 2000, 2000

RDC, PCS, CCR, TRDC, PCS, CCR, T11, CS and NOE , CS and NOE are consistent with one anotherare consistent with one another

All All rerestraints are included in straints are included in PARAMAGNETIC DYANAPARAMAGNETIC DYANA## (CYANA(CYANA##), and ), and

PARAPARArestraintsrestraints forfor XplorXplor--NIHNIH**

available atavailable at

www.postgenomicnmr.netwww.postgenomicnmr.net

AnAn integrated package to exploit integrated package to exploit paramagnetic paramagnetic rerestraintsstraints

#Güntert, Wütrich, J.Mol.Biol. 1991*Clore, Gronenborn, Brunger, Karplus, J.Mol.Biol. 1985; Schwieters, Kuszewski, Tjandra,Clore, J.Magn.Reson., 2003

Bertini, Luchinat, Parigi Progr. NMR Spectr. (2002) 40, 249-273Bertini, Luchinat, Parigi Concepts Magn. Reson., (2002) 14, 259-286Banci, Bertini, Cavallaro, Giachetti, Luchinat, Parigi, J. Biomol. NMR (2004) 28, 249-261Barbieri, Luchinat, Parigi, Chem. Phys. Chem. (2004) 5, 797-806.Bertini, Luchinat, Parigi, Pierattelli, ChemBioChem (2005) 6, 1536-1549

1212

Towards structureTowards structuress without NOEswithout NOEs

0 50 100 150 200 250 1800 20000

1

2

3

4

5

BB

RM

SD

(Å)

number of NOEs

Bertini, Donaire, Jiménez, Luchinat, Parigi, Piccioli, Poggi, J. Biomol. NMR 2001

Structure can be obtained with PCS, RDC, CCR and less

than 10 NOEs

+ 908 + 908 pcspcs CeCe, , YbYb, , Dy Dy +1703 +1703 pcspcs 11 lanthanides11 lanthanidesRMSD=1.28 RMSD=1.28 ÅÅ RMSD=0.82 RMSD=0.82 ÅÅ

17 hydrogen bonds, 105 dihedral angles, 17 hydrogen bonds, 105 dihedral angles, + 26 + 26 TT11 CeCe(III) (III)

+ 181 HN+ 181 HN--N N rdcrdc CeCe(III), (III), DyDy(III) and (III) and YbYb(III)(III)

Barbieri, Luchinat, Parigi, ChemPhysChem, 2004

Ca2-Oncomodulin

CaTb-Oncomodulin

Heteronuclear detection Heteronuclear detection minimizes minimizes line line broadening broadening and and signal losssignal loss

Babini, E.; Bertini, I.; Capozzi, F.; Felli, I.C.; Lelli, M.; Luchinat, C. J. Am.Chem. Soc. 2004, 126, 10496

1H-15N HSQC protonless 13CCOO13CCAA MQMQ

( )τωγ ,6

2

2,1 fr

kR I=

(γ13C)2 ~ (γ1H)2/16(γ15N)2 ~ (γ1H)2/100

55/109 peaks lost 37/109 peaks lost

1313

TbCa

Up to 11 Å

Up to 16 Å

TbTb--Oncomodulin: how Oncomodulin: how close toclose to the metal?the metal?

Up to 8 Å

Obtained on a special 175 MHz 13C direct high sensitivity probe

Visible in HSQC and COCA

Visible in COCA

Visible in NOESY

Visible in 13C 1D

Still Unassigned

Up to 5.5 Å

PCS and RDC, plus crystal structure, are used to calculatethe solution structure of complexes of CaM with targetpeptides from DAP kinases

Structural refinement in Structural refinement in CaMCaM complexescomplexes

in collaboration with Wilmanns@embl-hamburg.de

Calmodulin

CaM-binding domain

Xray structure

-35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30-35

-30

-25

-20

-15

-10

-5

0

5

10

15

20

25

30

3

4

5

6

7

8

9

10

1112

13

1415 16

17

1819

20

37

38

39

40

41

45

46

4750

76

7778

793

4

5

6 7

8

910

1112

13 14 1516

17 18 192021

22

2324

3132

33

34

3536

37

38

39 4041

4244

45

46

47

48

49

7273

74 7

8

9

10

11

12

13

14

15

1618

37

38

4041

4244

45

46

47 5078

Tb Tm Dy

RD

C o

bs (H

z)

RDC N-ter calc (Hz)

PCS provide the χ tensors

Exp. RDC deviate from predictions based on the X-ray

structure due to structural rearrangements in solution

Bertini, Luchinat, Parigi,Bertini, Luchinat, Parigi, Wilmanns et Wilmanns et al., al., submittedsubmitted

1414

Structural refinement in Structural refinement in CaMCaM complexescomplexes

-35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30-35-30-25-20-15-10-505

1015202530

Tm Dy Tb

RD

C o

bs (H

z)

RDC calc (Hz)

The solution structure is found significantly different from the solid state structure due to a different relative position of the two CaM domains

Even larger differences are being observed in other CaM-

peptide complexes

X-ray structure

NMR structure

NN

CC

A“true” solid state structure “true” solution structure

X-ray structure NMR structure

Indetermination ofthe X-ray structure

Indeterminationof the NMR structure

“true” solid state structure “true” solution structure

X-ray structure NMR structure

indetermination of the“NMR-corrected” X-ray structure

“NMR-corrected”X-ray structure

B

Can the Can the accuracyaccuracy of aof a protein structureprotein structure in in solution be extendedsolution be extended??

1515

A challenge for A challenge for paramagnetismparamagnetism--based restraints: based restraints: proteins with partially independent domainsproteins with partially independent domains

N-terminal

C-terminal

Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. & Bax, A Biochemistry(1992)

Baber, J. L., Szabo, A. & Tjandra, N. J. Am. Chem. Soc. (2001)

Small 15N-1H NOEsfor residues 78-81

The case of CaThe case of Ca44CalmodulinCalmodulin

A paramagnetic ion is attached to the N-terminal domain, causing its partial orientation in the magnetic field

The Florence strategyThe Florence strategy

The C-terminal domain MAY experience some induced orientation

BertiniBertini, , GelisGelis, , KatzarosKatzaros, Luchinat, , Luchinat, ProvenzaniProvenzani, Biochemistry, 2003, Biochemistry, 2003

Ln3+

N

C

B0

1616

- 3 0 -2 0 -1 0 0 1 0 2 0 3 0 -3 0 -2 0 -1 0 0 1 0 2 0 3 0

Tb3+ Tm3+

rdc of the N-terminal domain

RDC of NRDC of N-- and Cand C--terminal domainsterminal domains

- 3 0 -2 0 -1 0 0 1 0 2 0 3 0

Dy3+

- 6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6

rdc of the C-terminal domain

Tb3+ Tm3+ Dy3+

Ln3+

N

CDistribution of the rdc values in the two domains

Ln3+

N

C

BertiniBertini, Del , Del BiancoBianco, , GelisGelis, , KatsarosKatsaros, Luchinat, , Luchinat, ParigiParigi, , PeanaPeana, , ProvenzaniProvenzani, , ZorodduZoroddu, , Proc. Natl. Acad. Proc. Natl. Acad. SciSci. . USAUSA, 2004, 2004

PCS and RDC can be used together to obtain the maximum allowed probability (MAP) for each conformation

Conformations with largest MAP arethose in which the system can stay longer

MAP = maximum weight for a conformation independently of all other experienced conformations

Maximum Allowed Probability calculation Maximum Allowed Probability calculation for the different conformationsfor the different conformations

Bertini, Gupta, Luchinat, Parigi, Peana, Sgheri, Yuan, JACS, 2007

C

NLn3+

NLn3+

C

Longinetti, Luchinat, Parigi, Sgheri, Inverse Problems, 2006

1717

Conformational heterogeneityConformational heterogeneity in in calmodulincalmodulin

Conformations of calmodulin with largest MAPThe weight of each conformation cannot be larger than MAP (0.35)

Bertini, Luchinat, et al. PNAS, 2004Bertini, Gupta, Luchinat, Parigi, Peana, Sgheri, Yuan, JACS, 2007

N

linker

Structure Structure of calmodulin of calmodulin interactinginteracting withwith the the intrinsicallyintrinsically unfoldedunfolded proteinprotein αα--synucleinsynuclein

A

N CThe The structure structure of of each domain is very each domain is very similar to isolated calciumsimilar to isolated calcium calmodulin calmodulin

Chemical shift changes indicate that an interaction occurs

Refined using PCS

Refined using RDC

larger spreadinglarger spreading of Cof C--terminal rdcterminal rdc thanthan in thein theisolated proteinisolated protein

Titrations indicate Kd ~10-5 M

1818

-30 -20 -10 0 10 20 30 -30 -20 -10 0 10 20 30

-6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6

-6 -4 -2 0 2 4 6RDC (Hz)

-6 -4 -2 0 2 4 6RDC (Hz)

-6 -4 -2 0 2 4 6R D C (H z)

-30 -20 -10 0 10 20 30

RestrictionRestriction inin conformational heterogeneityconformational heterogeneity

NN--terminal terminal domaindomain

CC--terminal terminal domain domain in in αα--synucleinsynuclein adductadduct

CC--terminal terminal domain domain in in freefree CaMCaM

Tb3+ Tm3+ Dy3+

Larger spreading in rdc corresponds to a reduced orientational (conformational) freedom

Conformational heterogeneity Conformational heterogeneity in the in the calmodulincalmodulin--αα--synucleinsynuclein adductadduct

Largest MAP conformations of calmodulin in its adduct with α-synuclein

Such conformation resembles the “canonical” closed conformation of calmodulin, although MAP is only 0.35

BertiniBertini, , GuptaGupta, Luchinat, Parigi, Peana, , Luchinat, Parigi, Peana, SgheriSgheri, , YuanYuan, J. , J. AmAm. . ChemChem. . SocSoc, 2007, 2007

1919

ProteinProtein--Protein ComplexesProtein Complexes

PCS and RDC can provide the relative position of twoproteins in a complex.

If the adduct is not rigid, PCS and RDC are averaged on the values relative to all experienced conformations

Ln3+

RDC provide information on the orientations with respect to the metal tensor

PCS provide information on the positions with respect to the metal tensor

Assessing the usefulness of LanthanideAssessing the usefulness of Lanthanide--Binding Tags Binding Tags (LBT) to investigate protein(LBT) to investigate protein--protein interactionsprotein interactions

A tag able to bind paramagnetic metal ions can be exploited to obtain structural and dynamic informationA LBT tag has been fused with the copper chaperoneHAH1Theoretically, RDC are scaled down by mobility but still fully usable. PCS are scaled down and not fully usable

HAH1

LBT Ln3+

HAH1 MNK1

Er3+

HAH1 MNK1

Dy3+

MNK1? The PCS obtained by inserting Tm3+, Dy3+, Er3+, and Tb3+

on LBT have been tested for their ability to monitor the interaction of HAH1 with MNK1

2020

Flexibility in LBTFlexibility in LBT--HAH1 makes the solution not univocalHAH1 makes the solution not univocal

Only calculations starting with one of the two HAH1-G1LBT conformations resulted in a structure of the adduct that is similar to that expected:flexibility hampers the use of PCS

Second generation tags have been conceived to reduce flexibility:Su, Huber, Dixon, Otting, ChemBioChem, 2006, 7, 1599Martin, Hähnke, Nitz, Wöhnert, Silvaggi, Allen, Schwalbe, Imperiali, JACS, 2007, 129, 7106Keizers, Desreux, Overhand, Ubbink, JACS, 2007, 129, 9292…

HAH1 MNK1

- Solid state NMR structures are difficult to obtain because protons cannot be routinely observed and NOE-like cross peak intensities are not quantitatively related to distances

-- Paramagnetic proteins may provide distinct advantages:Paramagnetic proteins may provide distinct advantages:in SS NMR Curie relaxation is absent (no rotation!)PCS should be equally well observable…

• insoluble proteins• fibrils• membrane proteins

Solid state NMR of paramagnetic proteinsSolid state NMR of paramagnetic proteins

Why SS NMR of Proteins?Why SS NMR of Proteins?

Why SS NMR of Why SS NMR of paramagneticparamagnetic Proteins?Proteins?

2121

ZnCatalytic-active domain of the

Matrix MetalloProteinase 12

159 AA (17.6 kDa)Zn(II)-containing proteinZn(II) replaceable with Co(II)(Bertini et al., PNAS, 2005)

44 AA (28%) helices27 AA (17%) ß-strands

Easily crystallisable

A test case: Zinc(II)A test case: Zinc(II) and and Cobalt(II)Cobalt(II) MMPMMP--1212

Assigned with:

13C-13C PDSD (@700MHz)11.5 kHz MASτmix= 15ms

J-dec PDSD3D NCACX PDSD3D NCOCX PDSD

94% 13C bb90% 13C sc90% 15N bb

Balayssac, S.; Bertini, I.; Fälber, K.; Fragai, M.; Jehle, S.; Lelli, M.; Luchinat, C.; Oschkinat, H.; Yeo, K.J. ChemBioChem (2007), 8, 486 – 489

Catalytic domain of Catalytic domain of ZnMMPZnMMP--12 12 Crystalline (Crystalline (DiamagneticDiamagnetic) 18kDa) 18kDa

2222

Assigned with:

13C-13C PDSD (@700MHz)11.5 kHz MAS τmix= 60ms

J-dec PDSD2D NCA, 3D NCACX-PDSD2D NCO, 3D NCOCX-PDSD

Observed:85% aaof which70% aa assigned

Balayssac, S.; Bertini, I.; Lelli, M.; Luchinat, C.; Maletta, M. J. Am. Chem. Soc.(2007), 129, 2218–2219

Catalytic domain of Catalytic domain of CoMMPCoMMP--1212CrystallineCrystalline ((ParamagneticParamagnetic))

PCS

13C-13C PDSD 11.5kHz MASZnMMP-12 (Blue, Diamagnetic)

CoMMP-12 (Red, Paramagnetic)

CoMMPCoMMP--1212 vsvs ZnMMPZnMMP--1212

Balayssac, S.; Bertini, I.; Lelli, M.; Luchinat, C.; Maletta, M. J. Am. Chem. Soc.(2007), 129, 2218–2219

2323

-3

-2

-1

0

1

2

3

4

5

6

0 50 100 150 200 250

solid obssolid calcliquid obsliquid calc

PCS (ppm)

K151-A157

S230-A234

13C nuclei

SolutionSolution vsvs SolidSolid--State State PCSPCS

Balayssac, S.; Bertini, I.; Lelli, M.; Luchinat, C.; Maletta, M. J. Am. Chem. Soc.(2007), 129, 2218–2219

Intra-Molecular and Inter-Molecular PCSInteraction with all the Co2+ lattice ions

29 Å 35 Å

24 Å

20 Å

9.4 ÅA

9.6 Å

B

40 Å

30 Å

xy

z

A29 Å 35 Å

24 Å

20 Å

9.4 ÅA

9.6 Å

B

40 Å

30 Å

xy

z

xy

z

A

Ser 230

12.0 Å

Nearest Co2+ Atom

Bound Co2+ Atom

10.5 Å

CoMMPCoMMP--1212 Pseudo Contact Shifts:Pseudo Contact Shifts:IntraIntra-- andand InterInter--Molecular EffectsMolecular Effects

Balayssac, S.; Bertini, I.; Lelli, M.; Luchinat, C.; Maletta, M. J. Am. Chem. Soc.(2007), 129, 2218–2219

2424

13C-15N Co-MMP12

13C-15NZn-MMP12

Paramagnetic-diluted sample Reversed Paramagnetic-diluted

Inter PCSIntra PCS

Balayssac, S.; Bertini, I.; Bhaumik, A.; Lelli, M.; Luchinat, C. Submitted

ParamagneticParamagnetic--diluted samplesdiluted samples::Direct and Direct and Reverse DilutionReverse Dilution

Intra and intermolecular PCS can be separated

Bertini, I.; Bhaumik, A.; Lelli, M.; Luchinat, C. In preparation

Protein Structure with Structural Protein Structure with Structural Restraints fromRestraints from IntraIntra--Molecular SS PCSMolecular SS PCS

2525

Balayssac, S.; Bertini, I.; Bhaumik, A.; Lelli, M.; Luchinat, C. Submitted

Crystallographic positionsParamagnetic NMR positions

2.1Å0.3Å!

3.2 Å

SolidSolid--state Protein Arrangement state Protein Arrangement Determined with InterDetermined with Inter--Molecular SS PCSMolecular SS PCS

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