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Ca2 binding protein

• Substitution of Ca2 with Ln3
• Full assignment with standard techniques
• Direct detection 13C experiments

Identification of ligands to obtain new
structural constraints
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Observed Hyperfine Shift
Pseudocontact shift The program FANTASIAN1 can
predict pseudocontact shifts
Contact shifts Values of about 10 ppm for
backbone 13CO coordinating Ce are expected
Identification of backbone CO coordinating the
metal
L. Banci, I. Bertini, K.L. Bren, M.A. Cremonini,
H.B. Gray, C. Luchinat, P. Turano, JBIC 1996
Bertini, Lee, Luchinat, Piccioli, Poggi,
ChemBioChem 2001
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Nuclear relaxation due to the electron-nucleus
dipolar coupling Solomons equations
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Constraint surfaces
A
B

M
A
C
B
C
D
NOE
T1
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Residual Dipolar Coupling
Pseudocontact shift
Residual Dipolar Coupling
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PCS RDC CCR
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positive
Pseudocontact shifts
negative

Axial Totally Rhombic
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Pseudocontact shifts
 
A

A
M1, M2, M3
M
C
B
3 atoms, the same metal ion
Three metal ions, the same atom
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• Given ri, ?, ? and ??s, it is straightforward to
  find ?pc
• Given ri, ? and ? and ?pc, it is still
  straightforward to find ??s
• FANTASIAN and FANTALIN programs available
• at www.postgenomicnmr.net
• Given ?pc only, there is not a univocal set of
  the other parameters

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Pseudocontact shifts
M
The relative position of the metal ion with
respect to any rigid domain can be determined but
NOT the directions of the c tensor axes. PCS do
not provide a unique solution, unless two sets,
relative to two different metal ions, are used.
M
Bertini, Longinetti, Luchinat, Parigi, Sgheri, J.
Biomol. NMR 2002
The program PSEUDYANA is available at the web
site www.postgenomicnmr.net
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RDC
3 NH, the same metal ion
Two metal ions, the same NH
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Tensor 1
Tensor 2
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• Given ?, ? and ??s, it is straightforward to
  find Drdc
• Given ?, ? and Drdc, it is still straightforward
  to find ??s
• FANTORIENT program available at
• www.postgenomicnmr.net
• Given Drdc only, there is not a univocal set of
  the other parameters

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Curie-Dipole Dipole Cross Correlation
CCR
B0
M
q
time averaged electron magnetic moment ltmgt
N
H
Real part of the spectral density
Bertini, Luchinat, Tarchi, Chem. Phys. Lett.,
1993 Bertini, Luchinat, Piccioli, Tarchi Concept
Magn. Reson., 1994 Boisbouvier, Gans, Blackledge,
Brutscher, Marion, J.Am.Chem.Soc., 1999
Imaginary part of the spectral density
Ghose, Prestegard, J. Magn. Reson., 1997
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Curie-Dipole Dipole Cross Correlation
Effect of reducing J in an antiphase doublet
having equal / unequal linewidths
True and false COSY cross peaks
Bertini, Luchinat, Tarchi Chem. Phys. Lett., 1993
Bertini, Luchinat, Piccioli, Tarchi Concept
Magn. Reson., 1994
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Curie-Dipole Dipole Cross Correlation
Real part of the spectral density
(proposed by Marion et al. as structural
constraints) Boisbouvier, Gans, Blackledge,
Brutscher, Marion, J.Am.Chem.Soc., 1999
Imaginary part of the spectral density
Ghose, Prestegard, J. Magn. Reson., 1997
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Isotropic versus Anisotropic Curie-DD Cross
Correlation
Following Vega and Fiat, the dipolar shift
Hamiltonian is H gA IA?s?B0
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CCR
Complete theory for CCR outside spin Hamiltonian
formalism
The angles qXAK, qYAK and qZAK specify the
directions of the principal axes X, Y and Z of
the shift tensor of nucleus A with respect to the
AK axis. The principal axes for the shift tensor
are obtained by first calculating the tensor in
the principal frame of the susceptibility tensor
and then diagonalising the symmetric part
Bertini, Kowalewski, Luchinat, Parigi J. Magn.
Reson. 2001, in press
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Isotropic versus Anisotropic Curie-DD Cross
Correlation
CCR
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CCR
Curie-DD CCR measured on lanthanide-substituted
calbindin
Bleaney, 1972
cisoBleaney cisofit Ce 4.6 4.4 10-32
m3 Yb 14.4 16.6 10-32 m3
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How to measure CCR
Separation of cross correlation effects on the
(strong) multiplet components (J-resolved G
experiments, TROSY-based sequences)
Selection of the (weak) signal arising from the
relaxation-allowed coherence transfer
(quantitative G experiments)
Tessari, Vis, Boelens, Kaptein and Vuister, JACS,
1997 adapted Bertini, Cavallaro, Cosenza,
Kümmerle, Luchinat, Piccioli, Poggi, J. Biomol.
NMR (2002)
Boisbouvier, Gans, Blackledge, Brutscher, Marion,
JACS., 1999
CCR obtained with exponential fitting of data
CCR obtained with linear fitting of data
The sequence is shorter, therefore S/N is higher
Better fitting of the data
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Quick structure
Hyperfine based structural constraints
- Pseudocontact shifts
- Residual dipolar couplings
- Cross correlation
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Towards structure without NOEs
At least 2 lanthanide ions are needed Bertini,
Longinetti, Luchinat, Parigi, Sgheri, J. Biomol.
NMR 2002
Structure can be obtained with PCS, RDC, CCR and
less than 10 NOEs
Bertini, Donaire, Jiménez, Luchinat, Parigi,
Piccioli, Poggi, J. Biomol. NMR 2001
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Perspectives
M
site II
site I
M
CSI
X 4
N
C
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Perspectives structure without assignment
3 PCS 3 RDC relative to two metal ions for
each tetrahedrally arranged atoms centered on the
Ca atom of single amino acids
Cb
Ca
C
N
Ha
M
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Solution structure of Ce2-substituted N-terminal
fragment of calmodulin (TR1C)
PCS
380 pcs values
Magnetic Anisotropy Tensors of Ce2-TR1C in metal
binding sites I and II
site I site II ??ax ? 3? (m3 ? 1032) -1.52
?0.02 1.29?0.02 ??rh ? 3? (m3 ? 1032) -0.62
?0.03 0.59?0.04 Ce3 RMSD (Å) 0.29 0.42
Bentrop, Bertini, Cremonini, Forsén, Luchinat,
Malmendal, Biochemistry 36, 11605 (1997)
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Structure of Ce substituted Calbindin D9k
Ca2
Ce3
site II
site I
C-terminal
N-terminal
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Calbindin structure refinement with paramagnetic
constraints
Diamagnetic constraints

• 1793 NOEs
• 57 phi values
• 46 psi values
• 30 Hbonds
• 13 1D-NOE (RMSD0.69Å)

• Paramagnetic constraints
• 1164 pcs from 11 lanthanides
• 26 T1 values
• 64 rdc from Ce (RMSD0.26 Å)

Bertini, Donaire, Jiménez, Luchinat, Parigi,
Piccioli, Poggi, J.Biomol.NMR 2001
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Structure of Ce3 substituted Calbindin D9k
Bertini, Donaire, Jiménez, Luchinat, Parigi,
Piccioli, Poggi, J.Biomol.NMR 2001
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Allegrozzi, Bertini, Janik, Lee, Liu, Luchinat
J.Am.Chem.Soc., 122, 4154 (2000)
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RDC
Structure-independent cross-validation of
RDCs CaTmCbiso CaLuCbbic CaTmCbbic Barbieri,
Bertini, Lee, Luchinat, Velders, J.Biomol.NMR 2002
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RDC
PCS
c tensors from PCS RDCs from gt2 metal ions q
and f angles (assuming rigid structure) or RDCs
from gt5 metal ions complete model-free analysis

RDC calc
RDC obs
q calc
f calc
f obs
q obs
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Predicted/observed pattern of pseudocontact
shifts and rdc induced by lanthanide ions
Open squares Theoretical expectation Filled
circles Experimental values
Bleaney, J.Magn.Reson. 1972 Allegrozzi,
Bertini, Lee, Liu, Luchinat, JACS 2000
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Pseudocontact shift-based structural refinement
in lanthanide-binding proteins
6-15A, Ce
Allegrozzi, Bertini, Janik, Lee, Liu, Luchinat
J.Am.Chem.Soc, 2000
6A
9-23A, Yb
15-38A, Dy
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Rdcs obtained on proteins dissolved in orienting
media nicely complement rdcs from
lanthanide-induced self-orientation
as demonstrated by the rdc values themselves
TmCb in bicelles provides rdc sum of rdc of TmCb
in isotropic media rdc of LuCb in bicelles
LnTm
Barbieri, Bertini, Lee, Luchinat, Velders, J.
Biomol. NMR 2002
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Cross-correlation between Curie and N-H dipolar
coupling
M
N
q
Curie
Dipole-dipole
H
Bertini, Luchinat, Tarchi, Chem. Phys. Lett.,
1993 Bertini, Luchinat, Piccioli,
Tarchi, Concepts Magn. Reson. 1994
 
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Yb3 in a protozoan EF-hand calcium binding
protein
(A) Experimental and (B) simulated plots of
intensity profiles of the original and
pseudocontact shifted cross peaks of G15 and G122
(K3 1.55E4 K2 2.46E4). (C) Simulated
plots of the intensity profiles of original and
pseudocontact shifted cross peaks of G15 and G122
obtained by interchanging the values of K2 and
K3 values
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Superposition of four Ca2-binding loops of
EhCaBP (shown in blue) with the corresponding
loops of CaM (red) and TnC (dark green).