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Paramagnetic based constraints
Lucia Banci CERM University of Florence
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NMR of paramagnetic molecules
The presence of unpaired electrons has profound
effects on the NMR spectra
chemical shifts can have sizeable
contributions (hyperfine shift) relaxation
rates are enhanced
M
A
N
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Types of couplings between nuclear and electron
spins
contact (through bonds)
E ?Ac?Sz?
M
?
S(S1)/3kT
dipolar (through space)
E ??I?S (3cos2?-1) /rMN3
N
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The hyperfine shift
- contact
?cont ?Ac?Sz?
M
- pseudocontact
d (ppm)
A
N
d (ppm)
20
25
15
60
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The origin of pseudocontact shifts

? 0
B0

E ?Iz?(?)(3cos2? -1)
?

N
If ? is isotropic
1
?
(3cos2? -1) dcos? 0
?
-1
? 90
???
?
N
If ? is anisotropic
1
?
?(?)(3cos2? -1) dcos? ? 0
-1
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Nuclear relaxation
R1,2 ? E2f(?c, ?)
- Contact relaxation
? Ac2 f(?s, ?)
- Dipolar relaxation
? (1/rNM6 )f(?c, ?)
- Curie relaxation
? (?Sz?2/rNM6 )f(?r, ?)
S 2(S1)2B02 /rNM6
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Nuclear relaxation due to the electron-nucleus
dipolar coupling Solomons equations
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Nuclear relaxation induced by metal ions
Calculated at 800 MHz for a proton at 5 Å from
the metal
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Nuclear relaxation induced by metal ions
Fe3 L.S.
(S1/2)
Fe3 H.S.
(S5/2)
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Paramagnetic constraints turning disadvantages
into advantages
? T1, T2 Contact shifts PCS
RDC CC Curie-DD
T1, T2 Robust, reliable, correspond to long range
metal-nucleus NOEs
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T1, T2 constraints
M
T1,T2 from metal
NOEs among nuclei
The constraints have the same spherical symmetry
as NOEs (1/r6) They are applied as M-N NOE
constraints
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A Fe4S4 domain of the 2?Fe4S4?2 ferredoxin from
C. pasteurianum
Without R1 constraints
With R1 constraints
Bertini I., Donaire A., Luchinat C., Rosato A.,
Proteins 1997
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Paramagnetic constraints
T1, T2 ? Contact shifts PCS RDC
CC Curie-DD
Contact shifts Robust, reliable, correspond to 3J
values involving the metal ion
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e-
M
H
? ? cos2 ?
?
D
C
e-
M
H
? ? sin2 ?
?
D
C
? M-D-C-H
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Dihedral angle dependence of hyperfine shifts of
H? nuclei of iron-coordinated cysteines
a 10.3 b -2.2 c 3.9
? oxidized C. Pasterianum Fd oxidized C.
acidi urici Fd ? reduced C. vinosum HiPIP
? reduced E. halophila HiPIP I ? reduced E.
halophila HiPIP II O reduced E. vacuolata HiPIP
I ? oxidized B. schlegelii Fd
Bertini, Capozzi, Luchinat, Piccioli, Vila, JACS
1994
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Reduced HiPIP I from E. halophila

• Banci L., Bertini I., Eltis L.D., Felli
  I.C.,Kastrau D.H.W., Luchinat C., Piccioli M.,
  Pierattelli R., Smith M.,
• Eur. J. Biochem. 1994

• Bertini I., Donaire A., Eltis L.D., Felli I.C.,
  Luchinat C., Rosato A., Eur. J. Biochem. 1996

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Calculated vs. observed shifts of methyl protons
in bis-histidine cytochromes
? 1-CH3, ? 3-CH3, ? 5-CH3, ? 8-CH3
I. Bertini, C. Luchinat, G. Parigi, F.A. Walker,
JBIC 1999
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Inclusion of histidine planes orientation in
structure calculations
Banci, Bertini, Cavallaro, Luchinat
(submitted)
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The case of oxidized cytochrome b5
Banci, Bertini, Cavallaro, Luchinat (submitted)
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Paramagnetic constraints
T1, T2 Contact shifts ? PCS RDC
CC Curie-DD
Pseudocontact shifts
?zz
z
X
?
y
r
M
?yy
?xx
?
y
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Pseudocontact shifts in cytochrome b5
Arnesano, Banci, Bertini, Felli, Biochemistry
1998
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Structure calculations with pseudocontact shifts
Starting from random coil structures, the
dynamics on the dihedral angles is calculated at
decreasing temperatures as driven by the
pseudopotential constituted by all structural
constraints
PSEUDYANA A module of DYANA
ti tolerance wi weight
The magnetic susceptibility tensor parameters are
fitted during the structure calculations no
assumption is needed
Banci L., Bertini I., Gori Savellini G., Luchinat
C., Wüthrich K., Güntert P., J. Biomol. NMR 1998
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The impact of pseudocontact shifts on solution
structure determination
Without pseudocontact shifts
With pseudocontact shifts
Banci et al., Horse heart cyt c, Biochemistry,
1997
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Protocols for using pseudocontact shifts for
solution structure determination
PSEUDODYANA (Banci, Bertini, Cremonini, Gori
Savellini, Luchinat, Wüthrich, Güntert, J.
Biomol. NMR 1998) module for the torsion angle
dynamic program DYANA HOW TO GET
IT http//www.mol.biol.ethz.ch/wuthrich/software/
dyana
PCSHIFT (Banci, Bertini, Gori Savellini,
Romagnoli, Turano, Cremonini, Luchinat, Gray,
Proteins Struct., Funct., Genet.,
1997) Integrated module of the SANDER module of
the AMBER package HOW TO GET IT http//www.amber
.ucfs.edu/amber/ amber.htmlobtain
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Paramagnetic constraints

• T1, T2
• Contact shifts
• PCS
• RDC
• CC Curie-DD

Residual dipolar couplings
Partial orientation at high magnetic fields
Magnetic anisotropy (in paramagnetic molecules
can be very high)
Orienting agents
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Partial molecular orientation of cytochrome b5 at
high magnetic fields
Banci, Bertini, Huber, Luchinat, Rosato JACS 1998
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Magnetic susceptibility tensors in cyt b5
Metal contribution
Calculated
diamagnetic metal contributions molecular
magnetic susceptibility
Experimental
Diamagnetic contribution
Banci, Bertini, Huber, Luchinat, Rosato JACS 1998
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Magnetic susceptibility tensors in cyt b5
 
 
Banci, Bertini, Huber, Luchinat, Rosato JACS 1998
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NH vectors in the protein frame of Fe(III) cyt
b562
Arnesano, Banci, Bertini, Van der Wetering,
Czich, Kaptein, J. Biomol. NMR, 2000
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Magnetic susceptibility tensors in cyt b562
Metal contribution
Calculated
diamagnetic metal contributions molecular
magnetic susceptibility
Experimental
Diamagnetic contribution
Arnesano, Banci, Bertini, Van der Wetering,
Czich, Kaptein, J. Biomol. NMR, 2000
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Magnetic susceptibility tensors in cyt b562
 
 
Arnesano, Banci, Bertini, Van der Wetering,
Czich, Kaptein, J. Biomol. NMR, 2000
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NH and CH Drdcs
CH and NH
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Quick structure determination backbone
constraints only
NOE3JPCS
NH,CH Drdc
CSI
a3
a4
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Structure calculations
Comparison of the structure obtained with all
available constraints with those obtained with
backbone constraints only

NOE3JPCS

NH,CH Drdc
CSI