Paramagnetic NMR: a versatile tool in structural biology · Paramagnetic NMR: a versatile tool in...
Transcript of Paramagnetic NMR: a versatile tool in structural biology · Paramagnetic NMR: a versatile tool in...
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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
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BioBio--labslabs
LibraryLibrary
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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
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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
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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 [email protected]
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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