A caged lanthanide complex as a paramagnetic shift agent for protein NMR

Article abstract of DOI:10.1002/chem.200306019

A lanthanide complex, named CLaNP (caged lanthanide NMR probe) has been developed for the characterisation of proteins by paramagnetic NMR spectroscopy. The probe consists of a lanthanide chelated by a derivative of DTPA (diethylene-triaminepentaacetic acid) with two thiol reactive functional groups. The CLaNP molecule is attached to a protein by two engineered, surface-exposed, Cys residues in a bidentate manner. This drastically limits the dynamics of the metal relative to the protein and enables measurements of pseudocontact shifts. NMR spectroscopy experiments on a diamagnetic control and the crystal structure of the probe-protein complex demonstrate that the protein structure is not affected by probe attachment. The probe is able to induce pseudocontact shifts to at least 40 A from the metal and causes residual dipolar couplings due to alignment at a high magnetic field. The molecule exists in several isomeric forms with different paramagnetic tensors; this provides a fast way to obtain long-range distance restraints.

Full text of DOI:10.1002/chem.200306019

FULL PAPER
A Caged Lanthanide Complex as a Paramagnetic Shift Agent for Protein
NMR
[
a]
[b]
[b]
[c]
Miguel PrudÜncio, Jan Rohovec, Joop A. Peters, Elitza Tocheva,
[
c]
[c]
[d]
[d]
Martin J. Boulanger, Michael E. P. Murphy, Hermen-Jan Hupkes, Walter Kosters,
Antonietta Impagliazzo, and Marcellus Ubbink*
[
a]
[a]
Abstract:
A
lanthanide complex,
manner. This drastically limits the dy-
namics of the metal relative to the pro-
tein and enables measurements of
pseudocontact shifts. NMR spectrosco-
py experiments on a diamagnetic con-
trol and the crystal structure of the
probe-protein complex demonstrate
that the protein structure is not affect-
ed by probe attachment. The probe is
able to induce pseudocontact shifts to
at least 40 ä from the metal and causes
residual dipolar couplings due to align-
ment at a high magnetic field. The mol-
ecule exists in several isomeric forms
with different paramagnetic tensors;
this provides a fast way to obtain long-
range distance restraints.
named CLaNP (caged lanthanide
NMR probe) has been developed for
the characterisation of proteins by par-
amagnetic NMR spectroscopy. The
probe consists of a lanthanide chelated
by a derivative of DTPA (diethylene-
triaminepentaacetic acid) with two
thiol reactive functional groups. The
CLaNP molecule is attached to a pro-
tein by two engineered, surface-ex-
posed, Cys residues in a bidentate
Keywords: cage
compounds
¥
lanthanides ¥ NMR spectroscopy ¥
proteins ¥ pseudocontact shift
[1,2]
Introduction
NMR structure determination.
However, in recent years,
it has been demonstrated that paramagnetism can provide
useful structural information and, as a result, determination
of the solution structure of proteins containing paramagnetic
Unpaired electrons present in radicals and metal ions cause
paramagnetic effects that strongly influence NMR spectra.
For some time, paramagnetism was considered a nuisance in
[3,4]
metals is now a reality.
The presence of an unpaired elec-
tron can cause both broadening and shifts of the resonances
in the NMR spectrum. Line broadening is a consequence of
enhanced relaxation. To a good approximation, this effect is
isotropic and falls off with the sixth power of the distance
between the metal and the observed nucleus. It is, therefore,
a sensitive tool for distance measurements, but it is localised
[
a] Dr. M. PrudÜncio, A. Impagliazzo, Dr. M. Ubbink
Leiden Institute of Chemistry
Gorlaeus Laboratories, Leiden University
P.O. Box 9502, 2300 RA Leiden (The Netherlands)
Fax : (+31)71-5274-349
E-mail: m.ubbink@chem.leidenuniv.nl
[1]
to the immediate surroundings of the metal or radical. On
the other hand, line shifts are caused by two effects: a) con-
tact shifts are due to delocalisation of the unpaired electron
onto the nucleus and are only relevant for nuclei not more
than several bonds away from the metal and b) pseudocon-
tact shifts (PCS) result from the dipolar interaction between
the (time averaged) unpaired spin and the nucleus, and their
size depends on the anisotropy of the magnetic susceptibili-
[
b] Dr. J. Rohovec, Dr. J. A. Peters
Laboratory of Applied Organic Chemistry and Catalysis
Delft University of Technology, Julianalaan 136
2
628 BL Delft (The Netherlands)
[
c] E. Tocheva, Dr. M. J. Boulanger, Dr. M. E. P. Murphy
Department of Microbiology and Immunology
University of British Columbia, #300±6174
University Boulevard, Vancouver BC
V6T 1Z3 (Canada)
[1]
ty. The magnitude of PCS is described by the magnetic sus-
ceptibility tensor ([Eq. (1)] in the Experimental Section)
and falls off with the third power of the distance between
the metal and the observed nucleus. For strongly paramag-
netic metals with a high anisotropy, PCS can be observed
[
d] H.-J. Hupkes, Dr. W. Kosters
Leiden Institute of Advanced Computer Science
Leiden University, Niels Bohrweg 1
2
333 CA Leiden (The Netherlands)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author. This includes:
Table S1: Assignments of the H and N amide nuclei in Zn-pseudoa-
zurin. Supplementary experimental methods SM1-Tensor determina-
tion.
[5,6]
1
15
for nuclei at long distances from the metal,
used to provide restraints for structure determination.
Paramagnetic molecules with strongly anisotropic magnetic
which can be
[2,4]
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3
252 ± 3260
susceptibility also show some degree of spontaneous align-
ment at high magnetic fields; this provides an easy way to
obtain residual dipolar couplings.
The fact that there are at least eight bonds between the
lanthanide ion and the nuclei in the protein means that con-
tact shift effects are negligible. Relaxation-induced line
broadening is also negligible or limited to very few residues,
because the metal in the probe is located >6 ä away from
the nearest amide. Finally, the molecule is able to cause
spontaneous partial alignment of the protein at high mag-
netic fields; this enables residual dipolar couplings to be
measured. This newly designed probe is able to accommo-
date any lanthanide ion and can, in principle, be useful in
providing variable-range structural information about large
proteins and protein complexes.
[7]
To take advantage of these paramagnetic effects in dia-
magnetic molecules, various types of paramagnetic probe
molecules have been used. The first group consists of solu-
ble probes, which are not covalently attached to the mole-
[
8±14]
cule of interest, such as paramagnetic metals
xide spin labels.
and nitro-
[15±17]
The structural information provided by
this category of probes is limited by the fact that any ob-
served PCS are very small, due to averaging effects and by
the limited range of paramagnetic relaxation effects they
induce. The second group includes probes that are attached
to the molecules of interest by selective covalent or coordi-
[
18±21]
nation bonds, such as nitroxide spin labels,
divalent
Results and Discussion
[22±24]
[25]
[26]
cobalt
or manganese
ions and lanthanides (Ln).
Spin labels have a limited action radius and cause no PCS,
while Co requires a specific binding site and induces a
limited range of PCS .
Design of the ClaNP molecule: The design of a molecule
that can be used as an artificial paramagnetic probe, capable
of generating useful structural information about proteins,
requires a number of conditions to be met. The most impor-
tant of these are a) that the probe reacts with specific resi-
dues in the protein, thus enabling its attachment point to be
conveniently selected; b) that it has little flexibility with
regard to the protein to prevent averaging of the pseudocon-
tact effect; c) that it can accommodate a paramagnet capa-
ble of generating a useful range of paramagnetic effects and
d) that its attachment does not significantly alter the struc-
ture and stability of the protein.
2
+
Paramagnetic lanthanides, with their unpaired electrons
located in the 4f inner shell, are superior paramagnetic
probes, because a) a range of paramagnetic effects can be
expected with different lanthanides, b) similar ionic radii
and geometries are observed for complexes of different lan-
thanides and c) lanthanum(iii), lutetium(iii) and yttrium(iii)
can be used as diamagnetic analogues. These advantages
2
+
were demonstrated for the Ca -binding protein calbindin,
in which the calcium atom was replaced by various lantha-
[27±29]
nides.
Obviously, this application of lanthanides is limit-
Cys residues rarely occur on protein surfaces and thiol
chemistry can be used for specific attachment reactions.
DTPA is an excellent chelator of lanthanides, yielding a
soluble complex, which can easily be modified. For these
reasons, two thiol reactive functional groups were added to
DTPA (Figure 1 top) for the attachment to two engineered
Cys residues on a protein surface. Attachment of this modi-
fied DTPA±Ln complex in a bidentate manner is aimed at
drastically limiting its mobility relative to the protein.
ed to proteins that contain a metal site capable of accommo-
3
+
dating a Ln ion and assumes that metal replacement is
isomorphic. In addition, it results in the broadening of reso-
3
+
nances in the vicinity of the Ln ion due to relaxation ef-
fects.
Recently, various covalent paramagnetic tags have been
developed and inserted on a genetic level at one of the ter-
[30±33]
mini of the protein.
Their main purpose is to cause
3
+
spontaneous alignment to obtain residual dipolar couplings.
They are less useful for restraints based on PCS or relaxa-
tion, because of limited choice in location and the dynamics
of the probe relative to the protein.
DTPA-bis(amides) are known to bind Ln ions in a cage-
like, octadentate fashion through the three nitrogen atoms
of the diethylenetriamine backbone, three carboxylate
oxygen atoms, and two amide oxygen atoms; overall this
[35]
We have designed a molecule that can be used as a para-
magnetic probe without the limitations previously men-
tioned. The caged lanthanide NMR probe (CLaNP) can be
specifically attached to the surface of a protein where two
cysteine residues have been engineered at a convenient dis-
tance from each other. This probe constitutes, in effect, an
artificial paramagnetic centre that can be positioned specifi-
cally on an otherwise diamagnetic protein, without causing
significant structural changes. The CLaNP molecule binds to
the protein in a bidentate way, thus it has reduced mobility
and allows PCS to be measured. Although PCS have been
reported once by using a similar probe attached to a single
yields neutral complexes. In the case of the DTPA-bis(a-
mide) of S-(2-methylaminomethyl)methanesulfothioate
(MTS) described in this work, the introduced sulfothioate
groups are expected to be ꢀ10 ä from each other (Figure 1
middle). During the reaction with two Cys residues
(Figure 1 bottom), a sulphinic acid molecule leaves each of
the two functional groups on the probe molecule and the
protein±CLaNP complex is formed.
Lanthanides are the metals of choice, because of their su-
perior paramagnetic properties discussed above. However, a
disadvantage is their high coordination number, hence the
prevalence of isomeric forms. These complexes have four
chiral centres; the three nitrogen atoms of the diethylenetri-
[34]
protein residue,
it appears that generally the probe is
3
+
much too dynamic in such cases; this is in accord with the
fact that there are six rotatable bonds between the probe
and the backbone of the protein. Linking the probe at two
positions on the protein surface results in a cyclic compound
with severely reduced mobility.
amine moiety and the Ln ion. Consequently, they may
4
occur in up to 16 (=2 ) enantiomeric forms. Upon binding
of the probe molecule to the protein, the DTPA-bis(amide)
becomes part of a cyclic structure and a maximum number
of eight diastereomers with different exchange rates may be
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M. Ubbink et al.
FULL PAPER
tachment of the bidentate probe and to avoid intramolecular
sulphur bridge formation. From the numerous residue pairs
that fit these criteria, the E51C/E54C double mutant
(
dCPsaz) was selected and produced. Thiol determination
indicated an accessible thiol content of 1.7 (ꢁ0.2) for
dCPsaz after reduction with DTT. To confirm the accessibili-
ty of the thiol groups, the protein was reacted with MTS
(
the functional molecule without Ln±DTPA); this resulted
in an increase of the mass by 151 Da (ꢁ2) (from 13473 Da
to 13624 Da). This confirms the accessibility of the thiol
groups in both engineered Cys residues of dCPsaz, because
the expected mass increase after reaction with two MTS
molecules is 150 Da.
The reduced form of the protein was reacted with either
the Y-containing, diamagnetic probe or the Yb-containing,
paramagnetic probe and separated from a fraction of dimer-
ic protein present after reaction by gel filtration. Mass spec-
trometry results revealed a mass of 14068 Da (ꢁ2) for the
Y
diamagnetic complex (hereafter CLaNP±dCPsaz) and a
mass of 14152 Da (ꢁ2) for the paramagnetic one (hereafter
Yb
CLaNP±dCPsaz). Taking into account the mass of both
the unreacted diamagnetic and paramagnetic probes
(
755 Da and 839 Da, respectively), these results indicate
that both leaving groups are absent in the complexes, which
shows that each of the probes binds dCPsaz by its two reac-
tive arms. Complexes with masses corresponding to the
ClaNP molecule being attached through a single disulphide
bridge were not observed.
The effect of the mutations and probe binding on the
three-dimensional structure of the protein was investigated
1
5
1
by comparing the [ N, H]-HSQC spectra of the diamagnetic
Y
CLaNP±dCPsaz complex and of the wild-type ZnPsaz. A
1
1
diff
plot of the H-amide chemical shift difference ( H-d ) be-
tween the two spectra (Figure 2) shows that the overall
structure of ZnPsaz does not change significantly due to the
mutations and probe binding.
Figure 1. The probe molecule. Top; chemical structure of the bis(MTS)
derivative of DTPA. The dots represent the atoms involved in the coordi-
nation of the lanthanide ion. Middle; ball-and-stick representation of the
Ln-(bis(MTS)±DTPA) complex, with carbon, nitrogen and oxygen atoms
in white, light grey and dark grey, respectively. Sulphur atoms are repre-
sented by large black spheres and the water oxygen atom and Ln atoms
are shown as small and a large dark grey spheres, respectively. Bottom;
schematic representation of the reaction between the probe molecule
[
2 2 3 2
R(S O CH ) ] and two thiol groups on a protein, P.
envisaged; this has consequences for the paramagnetic ef-
fects (see below).
Construction of the CLaNP±protein complex: To character-
ise its paramagnetic properties, the CLaNP molecule was at-
tached to pseudoazurin (Psaz) from Alcaligenes faecalis S6,
a well studied copper-containing electron transfer protein,
which does not have any surface-exposed Cys residues. Its
size (14 kDa) allows easy NMR characterisation, whilst al-
lowing paramagnetic effects to be measured up to ꢀ40 ä
from the metal in the probe. To avoid any interference with
sulphur chemistry and to exclude undesired paramagnetic
effects, the copper in this protein was replaced by zinc, a
change that is known to have a negligible effect on the
1
diff
Figure 2. Chemical shift differences between amide protons ( H-d ) of
Y
wild-type Zn-Psaz and CLaNP±dCPsaz. Residues 46 to 57 (marked with
*
) are not observed in the spectrum.
1
The average H-amide chemical shift difference observed
is less than 0.01 ppm and the overall similarity of the two
structures is confirmed by X-ray crystallography results (see
below). It should be noted that the resonances correspond-
ing to Cys51 and Cys54 and residues in their immediate vi-
[36]
structure of type 1 Cu proteins.
The distance between the sulphur atoms in the two engi-
neered Cys residues was aimed to be 8±10 ä, to enable at-
Y
cinity are not observed in the spectrum of the CLaNP±
dCPsaz complex. The reason for the disappearance of these
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Paramagnetic Shift Agent
3252 ± 3260
signals is unclear, but it could be caused by line broadening
due to an exchange process, most likely within the probe
molecule.
phur linkers and parts of the cage structure (Figure 3
bottom).
The yttrium atom and the ClaNP were refined at 70% oc-
2
cupancy yielding an average B-factor of 68 ä . The arms of
X-ray structure: The crystal structure of dCPsaz complexed
with the Y-ClaNP probe (PDB entry 1PY0) clearly defines
the location of the Y-probe and the Psaz fold (Figure 3 top).
the probe are poorly defined in the electron density map
and the cage structure likely adopts multiple conformations
in the crystal. The probe arms are bent such that the cage
structure approaches the Psaz
peptide. The closest atom of the
main chain of Psaz to the yttri-
a
um atom is the C of Gly52, sit-
uated 6 ä away. The Y atom is
located at 6.0 ä and 8.3 ä from
the sulphur atoms of Cys51 and
Cys54, respectively and ~30 ä
from the zinc atom at the type I
site. Overall, there is no change
in the fold of the E51C/E54C-
ZnPsaz variant structure rela-
tive to the native Psaz structure
as revealed by an rms deviation
a
between the C chains of less
than 0.3 ä.
Paramagnetic effects: Binding
of the paramagnetic probe to
dCPsaz causes paramagnetism-
induced shifts in the resonances
of all amides, as shown by the
comparison of the HSQC (het-
eronuclear single-quantum co-
herence) spectra of the para-
Yb
magnetic
CLaNP±dCPsaz
complex with that of the dia-
magnetic control complex,
Y
CLaNP±dCPsaz (Figure 4).
Several shifted resonances
are observed for each residue
as a result of the ability of the
CLaNP molecule to isomerise.
Y
Figure 3. ClaNP±dCPsaz complex crystal structure. Top; the secondary structure elements are coloured as fol- Our results show that the vari-
lows: b strands in dark grey and a helices in light grey. The side chains of residues Cys51 and Cys54 and the ous observed isomers have dif-
CLaNP are detailed as sticks. The disulphide bridges between Cys51 and Cys54 to the probe are shown in
ferent paramagnetic anisotro-
black. The zinc and the yttrium atoms are depicted as light grey and dark grey spheres, respectively. Bottom; a
Y
[51]
pies and tensor orientations,
which result in several resonan-
ces for each amide group. The
resonances for an amide group
approximately lie on a line with
stereoview of the CLaNP region. A SigmaA
weighted difference electron density map of the probe is
shown, with residues Cys51 to Cys54 and the yttrium atom contoured at 2.5s. In both panels, atoms are shown
as follows: carbons are white, oxygens and nitrogens are grey and sulphur atoms are in black.
Following structure solution by molecular replacement
with an apo-pseudoazurin model, a difference map was com-
puted to locate metals absent in the coordinate set. The
highest positive peak in a difference map appeared at the
type I site and was modelled as a zinc atom with full occu-
pancy. The next two highest peaks were located in the sol-
vent channels of the crystal. One of these peaks is near the
molecular surface and was modelled as a sulphate anion and
the other, 6±8 ä from residues Cys51 and Cys54 was model-
led as a Y atom. In difference maps of the probe at the end
of refinement, density was present for the Y atom, the sul-
a slope of 1. The reason for this is that all PCS, defined in
ppm, are independent of the nature of the nucleus that ex-
periences the paramagnetic effect and depend only on its
position relative to the metal [Eq. (1) see later]. Amide pro-
tons and nitrogens of an amino acid residue are in close
proximity of each other and should, therefore, experience
similar PCS. Thus, the fact that the shifted peaks lie nearly
on a straight line with a slope of 1 represents evidence that
the shifts are caused by the pseudocontact effect (Figure 4
bottom). The magnitude of the observed PCS is clearly de-
pendent on the distance between the affected amide and the
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M. Ubbink et al.
FULL PAPER
and two of lower intensity; this
probably reflects preferred con-
formations of the CLaNP mole-
cule. A diamagnetic resonance
is also observed for every
amide, which corresponds to
the fraction of unlabelled
dCPsaz in the sample.
Measurements at 17.6 Tesla
(
750 MHz) of the coupling be-
1
15
tween amide H and N atoms
showed differences between the
paramagnetic and the diamag-
netic complexes of up to 10 Hz,
which
indicate
that
the
Yb
CLaNP±dCPsaz is partially
aligned, causing residual dipolar
couplings to be observed. Fur-
ther investigations on partial
alignment with other lantha-
nides are underway.
As expected, for none of the
amides that could be observed
in the diamagnetic complex,
significant line broadening was
observed in the paramagnetic
complex; this is in accord with
the relaxation effect being lim-
ited to the immediate surround-
ings of the lanthanide.
Tensor calculations: From a set
of PCS, the size and orientation
of the corresponding magnetic
susceptibility tensor can be de-
termined, which can then be
used for further structural char-
acterisation of a protein or pro-
[37]
tein complex.
However, the
presence of multiple paramag-
netically shifted resonances for
each of the observed amides
somewhat complicates tensor
determinations, since there is
no unequivocal way of mapping
the resonances of similar inten-
sity to each tensor. In order to
solve this problem, a systematic
1
5
1
Y
Yb
Figure 4. Overlay of the [ N, H]-HSQC spectra of the CLaNP±dCPsaz (red) and the CLaNP±dCPsaz evaluation of all possible com-
(
black) complexes. Top; the complete spectrum. The resonances of the 31 amides used as inputs for the deter-
binations of the three most in-
tense resonances was carried
out. The two less intense reso-
mination of the paramagnetic tensors and those of the 37 additional amides assigned with the aid of the deter-
mined tensors (see text) are connected by blue and green lines, respectively, with slope 1. Side-chain amide
resonances are connected by horizontal black lines. The numeric labels represent the assignments of the pro-
1
5
tein residues in the diamagnetic complex. The asterisks denote the peaks belonging to N-acetamide nances were not taken into ac-
1
5
(
CH
3 2
CO NH ), used as an internal reference. Bottom; the set of resonances for the amide of Ala25. D-dia- count in these calculations,
magnetic resonance; P
1
2 3 4 5
, P , P , P and P - paramagnetically shifted resonances.
since they correspond to minor
conformers of the CLaNP mol-
Yb atom, in accordance with the third power distance de-
pendence [Eq. (1) see later]. Up to five shifted resonances
are observed for each residue, three equally intense ones
ecule. The multiple resonances for each amide results in a
spectral overlap of peaks of some residues. However, the
slope ꢀ1 observed for the paramagnetically shifted resonan-
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Paramagnetic Shift Agent
3252 ± 3260
Table 1. Magnetic susceptibility tensor parameters.
tween observed and predicted PCS. The reasons for this dis-
crepancy are unclear, but it may reflect a structural differ-
ence between crystallographic and solution conditions with
regard to the position of the amide proton of this residue.
The tensors determined in this way were used to predict the
shifts of another 37 residues in the protein to aid in the as-
signment of their corresponding resonances. These are
shown in Figure 6 with open symbols (*).
It could be shown that the solutions obtained for the ten-
sors are not unique. However, the excellent agreement
found between the predicted and observed values for the
shifted resonances of the 37 amide protons that were not in-
cluded in the initial analysis clearly demonstrates that the
computed tensors are able to describe the paramagnetic
effect caused by the three main isomers of the CLaNP mole-
cule.
Dcax
Dcrh
ꢂ32
3
ꢂ32
3
t
[a]
t
[a]
t
[a]
[10 m ]
[10 m ]
x ±x’
y ±y’
z ±z’
tensor A
tensor B
tensor C
12.6
10.2
8.0
14.8
12.3
8.4
52.3
47.8
31.6
168.8
57.9
151.3
128.4
49.5
161.5
[
a] Angles in degrees between the x, y, and z axes of the tensors and the
arbitrarily defined reference system (see text). The Dc values and axes
orientations are defined in such a way that the relationship Dcax, Dcrh >0
always holds.
ces greatly reduces the assignment problem, because it links
all resonances from one amide (Figure 4). Assignment of
the paramagnetically shifted resonances can thus be ach-
ieved based on the assignments of the diamagnetic resonan-
ces and is facilitated by the presence of a fraction of unla-
belled protein in the sample of the paramagnetic complex.
Therefore, a subset of 31 unequivocally assigned amides in
this complex, each with three resonances, was selected for
tensor determination.
Distance restraints without tensors: The occurrence of mul-
tiple resonances for each amide, caused by the isomers of
the probe, offers the possibility to derive distance restraints
The number of possible com-
binations increases very rapidly
with the number of amides, n
nꢂ1
(
as 6 ). Thus, subsets of 10
1
H-amide shifts were used to
search all possible combinations
and to determine the best set of
three tensors to describe the
PCS. Then, the sets were com-
bined and further optimised by
using a genetic algorithm. The
best solutions for the three ten-
sors (Figure 5) are given in
Table 1.
The observed versus calculat-
ed shifts of the 31 amide pro-
tons used for tensor determina-
tions are plotted, for each
tensor, as solid symbols (*) in
Figure 6. Tyr74 is found to be a
Figure 5. Stereoview of the orientation of the magnetic susceptibility tensors for the three main isomers of the
CLaNP molecule projected onto the Y atom of the CLaNP±dCPsaz complex structure. The axes of tensors
A, B and C (see also Table 1) are indicated, as well as the reference frame (y’ and z’; x’ is pointing towards the
back). Carbon, oxygen and nitrogen atoms are shown in black, dark grey and light grey, respectively. The sul-
Yb
Y
consistent outlier in the fits be- phur atoms of the Cys residues and the Y atom are shown as large spheres.
Figure 6. Plots of observed versus predicted PCS for 31 amide protons used for tensor determination (*) and an additional 37 amide protons assigned
pred
obs
with the aid of the determined tensors (*). The solid lines represent PCS =PCS
.
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M. Ubbink et al.
FULL PAPER
without using the magnetic anisotropy tensors. This is illus-
trated in Figure 7. The largest positive PCS of the three in-
tense resonances observed for each amide proton is plotted
range even further. Conversely, the shift range can be tuned
down by choosing lanthanides with a lower ability to shift.
The occurrence of several isomers of the CLaNP is still a
drawback presented by this molecule and work to synthesise
new probes with less or no isomers is in progress. We have
demonstrated, however, that the magnetic anisotropy ten-
sors of the different isomers can be calculated, despite the
degeneracy problem. We showed that the multiple resonan-
ces for each amide also offer an easy and fast way to obtain
distance restraints.
The CLaNP molecule can also cause spontaneous align-
ment at high magnetic fields, which provides a convenient
way to obtain residual dipolar couplings. The multiple iso-
mers could be beneficial in this case, because they result in
multiple alignment orientations of a protein in one sample
and in one experiment.
Finally, we are investigating the potential of CLaNP as a
relaxation agent. It can be expected that CLaNP containing
gadolinium can provide distance restraints on the basis of
Figure 7. Estimation of distance restraints based on largest observed PCS
for 68 amide protons. The lines represent the best fit with the inverse
cubic power of the distance (c), the theoretical maximum (a) and
the lower 95% prediction interval of the fit (g). A maximum PCS of
3+
relaxation. Contrary to other lanthanides, Gd has a long
electronic relaxation time causing strong relaxation effects.
Due to the isotropic nature of paramagnetic relaxation, it is
expected that isomeric forms of the probe will be irrelevant
to this application.
0
.6 ppm yields a target distance of 17.5 ä and distance range of 16±22 ä.
against the distance between the proton and the Yb atom.
The best fit with the inverse cubic power of the distance is
shown as a solid line. The dashed line indicates the theoreti-
cal maximum (based on the largest tensor in Table 1) and
the lower 95% prediction interval of the fit is shown by the
dotted line. Given an observed PCS, a target distance and
distance range can be read off by using the solid, dotted and
dashed lines, respectively. For example, a maximum PCS of
The CLaNP molecule offers the possibility of attaching
3
+
magnetically anisotropic Ln ions to any protein, irrespec-
tive of its native ability to bind lanthanides. Thus, CLaNP
represents a generally useful tool to provide variable-range
distance restraints in structure determination of proteins
and protein complexes, in the same way as is now standard
in paramagnetic metal proteins.
0
1
.6 ppm yields a target of 17.5 ä and the distance range of
6±22 ä. Like with NOE-based distance restraints, the low
Experimental Section
precision of the target needs not be problematic, provided
sufficient restraints can be observed. Such PCS distance re-
straints could be useful in docking calculations of two pro-
teins, as was demonstrated before on the complex of plasto-
Synthesis of the probe molecule: To prepare the bisanhydride form of
DTPA, a suspension of DTPA (49 g, 125 mmol) in acetic anhydride
(
6
53 mL, 560 mmol) and dry pyridine (62 mL, 770 mmol) was heated at
58C, while stirring for 24 h. After cooling to room temperature, the pre-
[38,39]
cyanin and cytochrome f.
For this purpose, the position
cipitate formed was filtered off and then washed with diethyl ether. After
of the lanthanide can be modelled on the basis of the crystal
drying under vacuum, pure DTPA-bis(anhydride) was obtained (43 g,
Y
13
1
20 mmol, 96%). C NMR ([D
6
]DMSO): d=50.78, 51.72, 52.71, 54.73,
structure of CLaNP±dCPsaz, with the lanthanide in a plane
g
165.88, 171.82 ppm. The sodium salt of the bis(MTS) derivative of DTPA
was prepared by dissolving the HBr salt of MTS (25.9 mg, 0.11 mmol)
with two Cys sulphur atoms at 7.3 ä from the two S (Cys)
g
b
atoms with Ln/S (Cys)/C (Cys) angles at 1298.
(
Toronto Research Chemicals) in DMSO (0.5 mL). In this solution,
DTPA-bisanhydride (19.3 mg, 0.054 mmol) was dissolved by heating.
After cooling to room temperature, this solution was washed with
DMSO (0.1 mL) into N-methylmorpholine (33.2 mg). After standing for
Conclusion
2
h at room temperature and a night at ꢂ208C, the solvent and the N-
methylmorpholine were evaporated under vacuum (0.1 mbar). The glassy
solid was dissolved in a solution of Na CO (23.8 mg) in water (1.5 mL).
We have designed a paramagnetic molecule, CLaNP, which
can be attached specifically to a protein surface by using
two engineered Cys residues at 8±10 ä distance. This biden-
tate mode of attachment sufficiently reduces the mobility of
the Ln ion relative to the protein to allow measurement
of PCS. Both NMR chemical shift analysis and X-ray dif-
fraction indicate that the structure of the protein is essen-
tially unaffected by the attachment of the probe. This makes
CLaNP the first general NMR probe designed to generate
PCS. In the current example, shifts induced by Yb are ob-
served up to 40 ä away from the metal and lanthanides that
induce larger PCS, such as Dy, are expected to extend this
2
3
The solution was filtered and then freeze-dried. The Ln-containing probe
molecule was prepared by mixing equimolar amounts of the lanthanide
ion and the bis(MTS) derivative of DTPA.
3
+
Protein expression and purification: The pseudoazurin gene from Alcali-
genes faecalis S-6 was amplified from the pUB1 vector kindly provided
by Dr. Makoto Nishiyama at the University of Tokyo by using the for-
ward primer, ’A/AG-CTT G/CT-AGC GAA AAT ATC GAA GTT CAT
ATG CT 3’ and the reverse primer, 5’CAT C/TC-GAG TCA TTT AGC
GCT GGC GAT GAC 3’. The PCR product was digested by using the
restriction endonucleases HindIII and XhoI and cloned into the transfer
¾
vector pBluescript II SK- (Stratagene, La Jolla, CA). This was followed
by a second cloning step into the NheI and XhoI sites of the bacterial ex-
pression vector pET24c (Novagen Inc). From DNA sequence analysis of
3258
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3252 ± 3260

Paramagnetic Shift Agent
3252 ± 3260
the pEPsaz plasmid obtained in this way, it was confirmed that no muta-
tions were generated during PCR amplification. For cloning purposes,
extra Ala and Ser residues were introduced at the ꢂ1 and 0 positions, re-
spectively. The modifications encoding the E51C and E54C mutations
were introduced in a single step by site-directed mutagenesis following a
These conditions resulted in the growth of twinned crystals that were
characterised as belonging to space group P6 with cell dimensions a=
5
b=42.7 ä, c=116.9 ä. The crystals were soaked in a mother liquor with
glycerol (30%) as a cryoprotectant and were looped directly into a cryo-
stream at 100 K. X-ray data were collected to 2.0 ä resolution on a
MAR345 image plate system and a home laboratory X-ray source. The
¾
procedure based on Stratagene×s ExSite PCR-Based Site-Directed Mu-
[
41]
tagenesis Kit and by using the pEPsaz plasmid as a template. High level
expression yielding soluble protein was observed in rich medium, while
the mutant protein was found in inclusion bodies when cultured on mini-
data were processed with DENZO and corrected for twinning by using
[
42]
the program Detwin and a 0.30 twin fraction. The structure of wild-
[
43]
type Psaz from A. faecalis was used as the starting model for molecular
1
5
[44]
mal medium. Therefore, uniformly N-labelled dCPsaz was obtained
replacement with the program MOLREP following the removal of the
from culturing Escherichia coli BL21(DE3) cells in E. Coli-OD4 N
medium (from Silantes GmbH), containing kanamycin (50 mgmL ).
Cells were grown at 378C to OD600 ꢀ0.7, induced with IPTG (0.5mm)
and allowed to grow for a further 5 h at 308C. Following harvesting by
centrifugation, the cells were resuspended in a buffer (100mm Tris-HCl,
copper and all solvent atoms. Eight percent of the data (573 reflections)
ꢂ1
were set aside to calculate the free R factor. Refinement was accomplish-
[
45]
[46]
ed by using CNS (for water picking) and Refmac5. The final struc-
ture includes the recombinant Psaz peptide (residues Ala-1 to Ala-120),
a Zn atom at the type I site and the complete probe, this resulted in an
Rwork and an Rfree of 19.3% and 23.2%, respectively. Statistics of the data
processing and structure refinement are presented in Table 2.
2
pH 7.2 containing 0.5m NaCl and 1mm ZnCl ) and disrupted in a French
pressure cell in the presence of phenylmethylsulfonyl fluoride (PMSF)
ꢂ1
(
1mm) and DNase I (50 mgmL ). After dialysis against MES (10mm
2
pH 6.5) with ZnCl (1mm), dCPsaz was purified by separating the crude
extract in a cation exchange carboxymethyl cellulose (CM) column equi-
librated with MES (10mm pH 6.5) and eluted with a gradient of NaCl
Table 2. Crystallographic data collection and refinement statistics
Data collection
(
0±250mm) in the same buffer; this was followed by gel filtration on a
resolution [ä]
R merge
2.0 (2.07±2.0)
0.057 (0.343)
25.3 (5.4)
Superdex G75 column equilibrated with a sodium phosphate buffer
1
5
13
(
50mm, pH 7.0) containing NaCl (150mm). The wild-type N(/ C)-Zn-
{
I}/{s(I)}
pseudoazurin was purified as described above from cells grown in M9
1
5
13
completeness [%]
unique reflections collected
refinement statistics
working R factor
99.2 (99.1)
8144 (2616)
4
minimal medium containing NH Cl (and uniformly C labelled d-glu-
cose) as the sole nitrogen (and carbon) source(s).
Construction of the protein±CLaNP complex: To substitute zinc for
copper, wild-type pseudoazurin and E51C/E54C-pseudoazurin samples
were prepared by the addition of cyanide (100mm) to a concentrated
0.193
0.232
0.016
28.5
free R factor
rmsd bond length [ä]
ꢂ
protein solution followed by immediate removal of the CN ion on a de-
2
overall B factor [ä]
salting Superdex G25 column equilibrated with MES (10mm, pH 6.5)
2
containing ZnCl (1mm). Excess Zn was removed on a desalting PD10
column equilibrated with sodium phosphate (20mm, pH 7.0). Before the
reaction between dCPsaz and the lanthanide probe, protein dimers were
dissociated by incubating the protein with DTT (3.5mm) for an hour at
room temperature; this was followed by DTT removal on a desalting
PD10 column, equilibrated with degassed sodium phosphate (20mm
pH 7.0). Two molar equivalents of the probe molecule were added drop-
wise to a protein solution (10 mL) in a sodium phosphate buffer (20mm,
pH 7.0). The reaction proceeded overnight at 48C under semi-anaerobic
conditions. Monomeric protein was separated from the reaction mixture
and dimer formed on a Superdex G75 column equilibrated with sodium
phosphate buffer (50mm, pH 7.0), which contained NaCl (150mm). Ap-
proximately 60% of this fraction contains the complex formed between
Determination of the magnetic susceptibility tensors: Experimental pseu-
pc
docontact shifts (d ) were obtained from the difference between the
Yb
shifts observed for the paramagnetic ( CLaNP±dCPsaz) and the diamag-
Y
pc
netic ( CLaNP±dCPsaz) complexes, d =dobs (paramagnetic complex)
ꢂdobs (diamagnetic complex). The pseudocontact shift experienced by a
pc
i
nucleus i in a paramagnetic molecule, d
, is described by Equation 1,
ꢀ
ꢁ
1
2pri
3
2
d pi c ¼ 1
Dcaxð3 cos q
2
ꢂ1Þ þ Dcrhðsin q
2
cos2W
Þ
i
i
i
ð1Þ
3
i i i
in which r , q and W are polar coordinates of nucleus i with respect to
Ln
the protein and the lanthanide probe (dCPsaz± CLaNP). The remaining,
the principal axes system of the magnetic susceptibility tensor. The axial
and rhombic anisotropies, Dcax and Dcrh, respectively, are defined as
Dcax =czzꢂ0.5(cxx +cyy) and Dcrh =cxxꢂcyy, in which cxx, cyy and czz are the
principal components of the magnetic susceptibility tensor. The Euler ro-
tation matrix G(a,b,g) converts the arbitrarily defined, metal-centred,
molecular coordinate system (the reference system), with polar (Cartesi-
an) coordinates r,q’,W’ (x’, y’, z’), into the magnetic coordinate system
free, protein was used as an internal diamagnetic control to facilitate the
1
5
1
assignment of [ N, H]-HSQC peaks.
NMR spectroscopy: NMR samples of wild-type pseudoazurin contained
2
protein (1.5mm) in sodium phosphate (20mm), pH 7.0, 6% (v/v) D O.
Ln
Samples of dCPsaz± CLaNP contained protein (0.5±1mm) in the same
buffer. Measurements were performed on a Bruker Avance DMX600
1
5
1
spectrometer operating at 303 K. [ N, H]-HSQC spectra were obtained
t
t
t
t
t
r,q ,W (x , y , z ). The arbitrary coordinates, x’, y’, z’, were determined by
1
5
1
with spectral widths of 32 ppm ( N) and 12.6 ppm ( H). Data processing
was performed in AZARA (available from ftp://ftp.bio.cam.ac.uk/pub/
Y
using the X-ray crystal structure of the CLaNP±dCPsaz complex (PDB
[47]
entry 1PY0). For this analysis the program MOLMOL was used to add
1
15
azara). Assignments of the H and N amide nuclei in Zn-pseudoazurin
proton coordinates. The reference frame was defined as having its origin
on the lanthanide atom and applying the Euler rotation matrix in Table 3
(
see Table S1 in the Supporting Information) were obtained with a
1
5
13
N, C-labelled sample by using an HNCACB experiment, in conjunction
Y
to the coordinates of the atoms in the structure of the CLaNP±dCPsaz
+
with previous assignments of Cu -containing pseudoazurin (results to be
published elsewhere). Spectra were analysed by using the program
complex.
Yb
In the CLaNP±dCPsaz complex, 31 amides were assigned unequivocally
[
40]
×Ansig for Windows×.
with each amide showing three equally intense resonances (A, B and C)
Mass spectrometry: Protein mass determination was performed with a Q-
TOF1 mass spectrometer (MS) (Micromass, Manchester, UK), equipped
with an on-line nano-electrospray source. The samples were diluted to a
pc
and corresponding pseudocontact shifts, di;j (j=A,B,C), which represent
three isomers of the CLaNP molecule.
ꢂ1
concentration of 3 pmolmL in water/methanol/acetic acid 50:50:1 (v/v/v)
and introduced into the MS by flow injection analysis. Mass spectra were
recorded from m/z 50±2000. The protein mass was calculated from the
protein envelope by deconvolution with the MaxEnt software.
Table 3. Euler rotation matrix values for definition of the x’, y’, z’ axes
system
0.134
0.935
0.329
0.051
0.325
ꢂ0.944
ꢂ0.990
0.144
ꢂ0.004
Y
Crystallisation and structure determination: Crystals of the ClaNP±
dCPsaz complex were grown at 188C in a mother liquor, which consisted
of ammonium sulphate (2.4m) and sodium phosphate (50mm) at pH 6.5.
Chem. Eur. J. 2004, 10, 3252 ± 3260
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3259

M. Ubbink et al.
FULL PAPER
By using two subsets of 10 amides each, all permutations of A, B and C
were used to calculate magnetic anisotropy tensors by means of a modi-
[19] A. Ramos, G. Varani, J. Am. Chem. Soc. 1998, 120, 10992±10993.
[20] J. L. Battiste, G. Wagner, Biochemistry 2000, 39, 5355±5365.
[21] V. Gaponenko, J. W. Howarth, L. Columbus, G. Gasmi-Seabrook, J.
Yuan, W. L. Hubbell, P. R. Rosevear, Protein Sci. 2000, 9, 302±309.
[22] A. Lewin, J. P. Hill, R. Boetzel, T. Georgiuo, R. James, C. Klean-
thous, G. R. Moore, Inorg. Chim. Acta 2002, 331, 123±130.
[
48]
fied version of the programme FANTASIAN. The tensors were sorted
according to their capability to predict the input data and the best solu-
tions were used as starting values for optimisation by using all 31 data
[
49,50]
points with an evolutionary algorithm called differential evolution.
Details of this approach are given in the Supporting Information. The
software used in the calculations is available upon request.
[23] K. Tu, M. Gochin, J. Am. Chem. Soc. 1999, 121, 9276±9285.
[24] V. Gaponenko, A. S. Altieri, J. Li, R. A. Byrd, J. Biomol. NMR
2
002, 24, 143±148.
The final tensors were used to predict the shifts (A, B, C) of another
[
[
[
[
[
[
[
25] J. Iwahara, D. E. Anderson, E. C. Murphy, G. M. Clore, J. Am.
Chem. Soc. 2003, 125, 6634±6635.
26] J. T. Welch, W. R. Kearney, S. J. Franklin, Proc. Natl. Acad. Sci.
USA 2003, 100, 3725±3730.
27] I. Bertini, M. B. L. Janik, Y. M. Lee, C. Luchinat, A. Rosato, J. Am.
Chem. Soc. 2001, 123, 4181±4188.
28] R. Barbieri, I. Bertini, G. Cavallaro, Y. M. Lee, C. Luchinat, A.
Rosato, J. Am. Chem. Soc. 2002, 124, 5581±5587.
29] M. Allegrozzi, I. Bertini, S. N. Choi, Y. M. Lee, C. Luchinat, Eur. J.
Inorg. Chem. 2002, 2121±2127.
30] V. Gaponenko, A. Dvoretsky, C. Walsby, B. M. Hoffman, P. R. Rose-
vear, Biochemistry 2000, 39, 15217±15224.
31] J. Feeney, B. Birdsall, A. F. Bradbury, R. R. Biekofsky, P. M. Bayley,
J. Biomol. NMR 2001, 21, 41±48.
3
7 amides, which helped to assign these amides unequivocally. These data
points are indicated in Figure 6 with open symbols (*).
Acknowledgements
We would like to thank Prof. G. W. Canters for many useful discussions
and Prof. C. Luchinat for critical reading of the manuscript. Prof. I. Berti-
ni is acknowledged for proving the source code of FANTASIAN. This
work was supported by the Research Training Network ×Transient× (con-
tract number HPRN-CT-1999-00095) and a National Science and Engi-
neering Research Council Discovery Grant to MEPM. MP acknowledges
the financial support provided through the European Community×s
Human Potential Programme under contract HPRN-CT-1999-00095,
TRANSIENT, and a Marie Curie Fellowship (contract number HPMF-
CT-2000±00928). MEPM is supported by a Canadian Institutes of Health
Research Scholarship.
[
[
32] C. Ma, S. J. Opella, J. Magn. Reson. 2000, 146, 381±384.
33] J. Wohnert, K. J. Franz, M. Nitz, B. Imperiali, H. Schwalbe, J. Am.
Chem. Soc. 2003, 125, 13338±13339.
[
34] A. Dvoretsky, V. Gaponenko, P. R. Rosevear, FEBS Lett. 2002, 528,
1
89±192.
[
35] C. F. G. C. Geraldes, A. M. Urbano, M. A. Hoefnagel, J. A. Peters,
Inorg. Chem. 1993, 32, 2426±2432.
[
[
[
1] I. Bertini, C. Luchinat, G. Parigi, Solution NMR of Paramagnetic
Molecules, 2001, Elsevier, Amsterdam.
2] I. Bertini, A. Rosato, P. Turano, Pure Appl. Chem. 1999, 71, 1717±
[36] H. Nar, R. Huber, A. Messerschmidt, A. C. Filippou, M. Barth, M.
Jaquinod, M. Vandekamp, G. W. Canters, Eur. J. Biochem. 1992, 205,
1123±1129.
[37] M. Gochin, H. Roder, Protein Sci. 1995, 4, 296±305.
[38] M. Ubbink, M. Ejdeback, B. G. Karlsson, D. S. Bendall, Structure
1998, 6, 323±335.
1
725.
3] L. Banci, I. Bertini, L. D. Eltis, I. C. Felli, D. H. W. Kastrau, C. Lu-
chinat, M. Piccioli, R. Pierattelli, M. Smith, Eur. J. Biochem. 1994,
2
25, 715±725.
[39] P. B. Crowley, G. Otting, B. G. Schlarb-Ridley, G. W. Canters, M.
Ubbink, J. Am. Chem. Soc. 2001, 123, 10444±10453.
[40] M. Helgstrand, P. Kraulis, P. Allard, T. H‰rd, J. Biomol. NMR 2000,
18, 329±336.
[41] Z. Otwinowski, W. Minor, Methods Enzymol. 1997, 276, 307±326.
[42] T. O. Yeates, Methods Enzymol. 1997, 276, 344±358.
[43] K. Petratos, Z. Dauter, K. S. Wilson, Acta Crystallogr. Sect.B. 1988,
44, 628±636.
[44] A. Vagin, A. Teplyakov, J. Appl. Crystallogr. 1997, 30, 1022±1025.
[45] A. T. Brunger, P. D. Adams, G. M. Clore, W. L. Delano, P. Gros,
R. W. Grosse-Kunstleve, J. S. Jiang, J. Kuszewski, M. Nilges, N. S.
Pannu, R. J. Read, L. M. Rice, T. Simonson, G. L. Warren, Acta
Crystallogr. Sect. D 1998, 54, 905±921.
[
[
[
[
[
[
4] I. Bertini, A. Donaire, B. Jimÿnez, C. Luchinat, G. Parigi, M. Piccio-
li, L. Poggi, J. Biomol. NMR 2001, 21, 85±98.
5] I. Bertini, M. B. L. Janik, Y. M. Lee, C. Luchinat, A. Rosato, J. Am.
Chem. Soc. 2001, 123, 4181±4188.
6] M. Allegrozzi, I. Bertini, M. B. L. Janik, Y. M. Lee, G. H. Lin, C. Lu-
chinat, J. Am. Chem. Soc. 2000, 122, 4154±4161.
7] J. R. Tolman, J. M. Flanagan, M. A. Kennedy, J. H. Prestegard, Proc.
Natl. Acad. Sci. USA 1995, 92, 9279±9283.
8] L. R. Dick, C. F. G. C. Geraldes, A. D. Sherry, C. W. Gray, D. M.
Gray, Biochemistry 1989, 28, 7896±7904.
9] G. Veglia, S. J. Opella, J. Am. Chem. Soc. 2000, 122, 11733±11734.
10] M. Sattler, S. W. Fesik, J. Am. Chem. Soc. 1997, 119, 7885±7886.
11] E. Liepinsh, M. Baryshev, A. Sharipo, M. Ingelman-Sundberg, G.
Otting, S. Mkrtchian, Structure 2001, 9, 457±471.
[
[
[46] G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta Crystallogr. Sect.
D 1997, 53 240±255.
[
[
12] S. Arumugam, C. L. Hemme, N. Yoshida, K. Suzuki, H. Nagase, M.
Bejanskii, B. Wu, S. R. Van Doren, Biochemistry 1998, 37, 9650±
[47] R. Koradi, M. Billeter, K. W¸thrich, J. Mol. Graphics 1996, 14 51±
55.
[48] L. Banci, I. Bertini, G. G. Savellini, A. Romagnoli, P. Turano, M. A.
Cremonini, C. Luchinat, H. B. Gray, Proteins: Struct. Funct. Genet.
1997, 29, 68±76.
9
657.
13] S. Aime, N. D×Amelio, M. Fragai, Y. M. Lee, C. Luchinat, E. Terre-
no, G. Valensin, J. Biol. Inorg. Chem. 2002, 7, 617±622.
[
[
14] G. Pintacuda, G. Otting, J. Am. Chem. Soc. 2002, 124, 372±373.
15] A. M. Petros, L. Mueller, K. D. Kopple, Biochemistry 1990, 29,
[49] R. Storn, IEEE Trans. Evol. Comput. 1999, 3 22±34.
[50] R. Storn, K. Price, J.Global Optimiz. 1997, 11 341±359.
[51] R. J. Read, Acta Crystallogr. Sect. A 1986, 42 140±149.
1
0041±10048.
[
16] M. Scarselli, A. Bernini, C. Segoni, H. Molinari, G. Esposito, A. M.
Lesk, F. Laschi, P. Temussi, N. Niccolai, J. Biomol. NMR 1999, 15,
1
25±133.
17] T. Yuan, H. Ouyang, H. J. Vogel, J. Biol. Chem. 1999, 274, 8411±
420.
18] S. U. Dunham, S. U. Dunham, C. J. Turner, S. J. Lippard, J.Am.-
Chem.Soc. 1998, 120, 5395±5406.
[
[
8
Received: December 12, 2003
Published online: May 6, 2004
3260
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3252 ± 3260