DOTA-M8: An Extremely Rigid, High-Affinity Lanthanide
Chelating Tag for PCS NMR Spectroscopy
Daniel Ha¨ussinger,*,† Jie-rong Huang,‡ and Stephan Grzesiek*,‡
Department of Chemistry, UniVersity of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland,
and DiVision of Structural Biology, Biozentrum, UniVersity of Basel, Klingelbergstrasse 50/70,
4056 Basel, Switzerland
Received April 22, 2009; E-mail: daniel.haeussinger@unibas.ch; stephan.grzesiek@unibas.ch
Abstract: A new lanthanide chelating tag (M8) for paramagnetic labeling of biomolecules is presented,
which is based on an eight-fold, stereoselectively methyl-substituted DOTA that can be covalently linked
to the host molecule by a single disulfide bond. The steric overcrowding of the DOTA scaffold leads to an
extremely rigid, kinetically and chemically inert lanthanide chelator. Its steric bulk restricts the motion of
the tag relative to the host molecule. These properties result in very large pseudocontact shifts (>5 ppm)
and residual dipolar couplings (>20 Hz) for Dy-M8 linked to ubiquitin, which are unprecedented for a small,
single-point-attachment tag. Such large pseudocontact shifts should be well detectable even for larger
proteins and distances beyond ∼50 Å. Due to its exceptionally high stability and lanthanide affinity M8 can
be used under extreme chemical or physical conditions, such as those applied for protein denaturation, or
when it is undesirable that buffer or protein react with excess lanthanide ions.
Introduction
Accurate determination of three-dimensional structures of
proteins in solution is one of the strongholds of modern NMR
spectroscopy. An even more demanding task is the precise
description of interaction sites and mechanisms in protein-protein
and protein-ligand complexes. All of these endeavors benefit
tremendously from recent developments in NMR techniques to
obtain long-range structural information. In addition to residual
dipolar couplings (RDCs), paramagnetic relaxation enhancement
(PRE) and pseudocontact shifts (PCS) caused by the interaction
of unpaired electrons in transition metal ions and nuclear spins
have recently attracted much attention.1-5
Due to their 1/r3 distance dependence, PCS yield particularly
valuable long-range distance and angular information. In many
cases, basic, very sensitive 2D-NMR experiments are sufficient
to extract precise PCS data, and as a consequence, PCS
determination is possible even for large biopolymers at the size
limit of current solution NMR techniques.5 To generate the PCS
effect, preferably lanthanide ions are attached to the protein,
since these transition metals have a particularly large magnetic
anisotropy. For the specialized case of metal-binding proteins,
this can easily be performed by exchanging the native metal
with lanthanide ions.6 In the absence of such a natural binding
site, the attachment has turned out to be quite a severe
stereochemical and physicochemical challenge, because lan-
thanides prefer a nine-fold coordination sphere, giving rise to
isomer formation.7-10 In addition, the metal ions need to be
positioned rigidly with respect to the protein; otherwise the PCS
is drastically reduced due to motional averaging. Several
lanthanide chelating tags (LCTs) based on EDTA,10-12 DTPA,7
and DOTA9,13 have been described that use convenient binding
to a single free cysteine on the surface of the protein. However,
thus far the observed PCS for these LCTs were much smaller
than for native metal-binding proteins, presumably due to the
motional freedom within the tag and/or of the tag relative to
the protein. Other approaches used zinc finger14 or EF hand15
domains fused to the protein of interest, which resulted in
similarly modest PCS and RDCs. More efficient tags have been
found in artificial lanthanide-binding peptides16-19 and in a rigid
† Department of Chemistry, University of Basel.
‡ Division of Structural Biology, Biozentrum, University of Basel.
(1) Bertini, I.; Luchinat, C. Curr. Opin. Chem. Biolo. 1999, 3, 145–151.
(2) Rodriguez-Castaneda, F.; Haberz, P.; Leonov, A.; Griesinger, C. Magn.
Reson. Chem. 2006, 44, S10–6, Spec No.
(3) Mittag, T.; Forman-Kay, J. D. Curr. Opin. Struct. Biol. 2007, 17, 3–
14.
(4) Pintacuda, G.; John, M.; Su, X. C.; Otting, G. Acc. Chem. Res. 2007,
40, 206–212.
(5) Otting, G. J. Biomol. NMR 2008, 42, 1–9.
(6) Allegrozzi, M.; Bertini, I.; Janik, M. B. L.; Lee, Y. M.; Lin, G. H.;
Luchinat, C. J. Am. Chem. Soc. 2000, 122, 4154–4161.
(7) Prudencio, M.; Rohovec, J.; Peters, J. A.; Tocheva, E.; Boulanger,
M. J.; Murphy, M. E. P.; Hupkes, H. J.; Kosters, W.; Impagliazzo,
A.; Ubbink, M. Chem.sEur. J. 2004, 10, 3252–3260.
(8) Vlasie, M. D.; Comuzzi, C.; van den Nieuwendijk, A. M. C. H.;
Prudencio, M.; Overhand, M.; Ubbink, M. Chem.sEur. J. 2007, 13,
1715–1723.
(9) Keizers, P. H.; Desreux, J. F.; Overhand, M.; Ubbink, M. J. Am. Chem.
Soc. 2007, 129, 9292–3.
(10) Ikegami, T.; Verdier, L.; Sakhaii, P.; Grimme, S.; Pescatore, B.;
Saxena, K.; Fiebig, K. M.; Griesinger, C. J. Biomol. NMR 2004, 29,
339–349.
(11) Gaponenko, V.; Altieri, A. S.; Li, J.; Byrd, R. A. J. Biomol. NMR
2002, 24, 143–148.
(12) Gaponenko, V.; Sarma, S. P.; Altieri, A. S.; Horita, D. A.; Li, J.; Byrd,
R. A. J. Biomol. NMR 2004, 28, 205–212.
(13) Ha¨ussinger, D. In Book of Abstracts; First European Conference on
Chemistry for Life Sciences, Rimini, Italy, October 4-8, 2005.
(14) Gaponenko, V.; Dvoretsky, A.; Walsby, C.; Hoffman, B. M.; Rosevear,
P. R. Biochemistry 2000, 39, 15217–24.
(15) Ma, C.; Opella, S. J. J. Magn. Reson. 2000, 146, 381–384.
(16) Wohnert, J.; Franz, K. J.; Nitz, M.; Imperiali, B.; Schwalbe, H. J. Am.
Chem. Soc. 2003, 125, 13338–13339.
Published on Web 09/28/2009
10.1021/ja903233w CCC: $40.75  2009 American Chemical Society J. AM. CHEM. SOC. 2009, 131, 14761–14767 9 14761

chemical tag based on dipicolinic acid,20 a tridentate ligand.
Lanthanide-loaded tags of this type resulted in large PCS and
RDCs, which are comparable to metal-binding proteins. The
drawback of these tags is their relatively low lanthanide affinity
(typically on the order of 10-6 to 10-8 molL-1), requiring
considerable excess of lanthanide ions in the NMR samples.19,20
Another very promising approach was demonstrated recently
by the Ubbink group, where a lanthanide chelator based on
DOTA8 or a cyclen-bis-pyridine oxide9 is attached to the target
protein via two amido-disulfide bridges. This results in very
rigid attachment, high affinity toward the metal, and very large
PCS and RDC effects. The drawback of this method is the need
for two solvent-accessible cysteine residues at a specific distance
from each other, which usually requires structural knowledge
beforehand and may also lead to unwanted disulfide formation
in certain cases.
Because of its extremely high affinity toward lanthanides on
the order of 10-25 to 10-27 molL-1,21 the cyclen derivative
DOTA (1,4,7,10 tetraaza-cyclododecane-tetraacetic acid) is
commonly used for caging lanthanides and other heavy metal
ions in magnetic resonance imaging and medical radioactive
applications. This high affinity also makes it an ideal lanthanide
chelating tag (LCT) for protein applications. Its extended polar
and hydrophobic surfaces are expected to engage in interactions
with the protein surface, thereby restraining its relative motion.
In this study we describe the syntheses of several new DOTA-
based LCTs. The particularly strong power of an eight-fold,
stereospecifically methylated DOTA (M8) to induce PCS is
demonstrated for two ubiquitin mutants in the folded state.
Furthermore, M8 can also be used to induce PCS in the unfolded
state under harsh conditions of denaturation.
Results
Syntheses of Tags, Metalation and Ligation to Proteins.
Starting from a commercially available precursor, we initially
synthesized a DOTA-based chelating tag (DOTA-SPy)13 con-
nected to the protein by a covalent disulfide bond (see formula
in Figure 1A). However, the dysprosium complex of this new
tag [Dy(DOTA-SPy)] resulted in rather moderate PCS of only
a few hundred ppb and correspondingly low RDCs of maximally
5 Hz when covalently linked to the ubiquitin mutant S57C
(Supporting Information, Figure S1). We reasoned that the small
paramagnetic effects were related to the internal mobility of
the metal chelator. This internal mobility became evident from
the temperature dependence of the 1H NMR spectrum of the
diamagnetic lutetium complex [Lu(DOTA-SPy)] (Figure 1A).
At 280 K, a complex spectrum is obtained corresponding to
the broken four-fold symmetry of the DOTA framework.
However, heating to slightly elevated temperatures leads to
coalescence of the spectrum at 333 K. Besides this internal
mobility, the flexibility of the linker between the protein and
the cyclen ring may also contribute to the partial averaging of
PCS and RDCs. The stereochemistry of DOTA is very
complex,22-26 and simultaneous motions of the four fused five-
membered rings (δδδδ or λλλλ) and of the acetic acid side arms
can yield two stereochemically nonequivalent configurations (∆
or Λ) around the metal center. When the metal-loaded tag is
covalently linked to a chiral molecule, e.g. a protein, a total of
four magnetically inequivalent stereoisomers can be formed. For
some residues in ubiquitin S57C tagged with [Dy(DOTA)] up
to three resonances were observed. Therefore, the interpretation
of the data and also the sensitivity of the experiments are
problematic, and DOTA-SPy appears of little practical use. A
similar stereochemical averaging problem is expected to affect
previously described LCTs based on EDTA and DTPA.
To avoid averaging of the PCS from different conformations,
we have followed a recently described route27,28 to rigidify the
(17) Martin, L. J.; Hahnke, M. J.; Nitz, M.; Wohnert, J.; Silvaggi, N. R.;
Allen, K. N.; Schwalbe, H.; Imperiali, B. J. Am. Chem. Soc. 2007,
129, 7106–7113.
(18) Su, X. C.; McAndrew, K.; Huber, T.; Otting, G. J. Am. Chem. Soc.
2008, 130, 1681–7.
(19) Su, X. C.; Huber, T.; Dixon, N. E.; Otting, G. ChemBioChem 2006,
7, 1599–604.
(20) Su, X. C.; Man, B.; Beeren, S.; Liang, H.; Simonsen, S.; Schmitz, C.;
Huber, T.; Messerle, B. A.; Otting, G. J. Am. Chem. Soc. 2008, 130,
10486–7.
(21) Rocklage, S. M.; Watson, A. D. J. Magn. Reson. Imaging 1993, 3,
167–78.
(22) Csajbok, E.; Baranyai, Z.; Banyai, I.; Brucher, E.; Kiraly, R.; Muller-
Fahrnow, A.; Platzek, J.; Raduchel, B.; Schafer, M. Inorg. Chem. 2003,
42, 2342–9.
(23) Csajbok, E.; Banyai, I.; Brucher, E. Dalton Trans. 2004, 2152–6.
(24) Aime, S.; Barge, A.; Botta, M.; Fasano, M.; Ayala, J. D.; Bombieri,
G. Inorg. Chim. Acta 1996, 246, 423–429.
(25) Aime, S.; Botta, M.; Fasano, M.; Marques, M. P. M.; Geraldes,
C. F. G. C.; Pubanz, D.; Merbach, A. E. Inorg. Chem. 1997, 36, 2059–
2068.
(26) Aime, S.; Barge, A.; Borel, A.; Botta, M.; Chemerisov, S.; Merbach,
A. E.; Muller, U.; Pubanz, D. Inorg. Chem. 1997, 36, 5104–5112.
(27) Ranganathan, R. S.; Pillai, R. K.; Raju, N.; Fan, H.; Nguyen, H.;
Tweedle, M. F.; Desreux, J. F.; Jacques, V. Inorg. Chem. 2002, 41,
6846–55.
Figure 1. Part of the aliphatic region of the 1H NMR spectra (14.1 T) of
(A) [Lu(DOTA-SPy)] at 333 K and at 280 K and (B) [Lu(M8-SPy)] at 333
K and at 280 K.
14762 J. AM. CHEM. SOC. 9 VOL. 131, NO. 41, 2009
A R T I C L E S Ha¨ussinger et al.