Detection of phosphorylation states by intermolecular sensitization of lanthanide-peptide conjugates

Article abstract of DOI:10.1039/c2cc34958b

The luminescence of a designed peptide equipped with a coordinatively- unsaturated lanthanide complex is modulated by the phosphorylation state of a serine residue in the sequence. While the phosphorylated state is weakly emissive, even in the presence of an external antenna, removal of the phosphate allows coordination of the sensitizer to the metal, yielding a highly emissive supramolecular complex.

Full text of DOI:10.1039/c2cc34958b

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COMMUNICATION
Detection of phosphorylation states by intermolecular sensitization of
lanthanide–peptide conjugatesw
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Elena Pazos, Marko Golicnik, Jose L. Mascarenas* and M. Eugenio Vazquez*
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Received 11th July 2012, Accepted 1st August 2012
DOI: 10.1039/c2cc34958b
The luminescence of a designed peptide equipped with a
coordinatively-unsaturated lanthanide complex is modulated by
the phosphorylation state of a serine residue in the sequence.
While the phosphorylated state is weakly emissive, even in the
presence of an external antenna, removal of the phosphate allows
coordination of the sensitizer to the metal, yielding a highly
emissive supramolecular complex.
Trp residue acting as a sensitizer and a terbium complex
attached to the peptide chain through Asp (1, 1P) or Glu
(2, 2P) side chains.14 The heptacoordinating DO3A (1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid), selected as
a
macrocyclic ligand, does not satisfy all the available coordina-
tion positions of the lanthanide ion,15 and therefore in the
unphosphorylated state the unfilled positions should be
occupied with water molecules, which are known to deactivate
the luminescence from the metal ion. Phosphorylation of the
serine residue should promote the intramolecular displace-
ment of the water by the pSer side chain phosphate group,
thus leading to an increased emission from the lanthanide ion
(Fig. 1).16,17
Phosphorylation is the fundamental signaling mechanism by
which eukaryotic cells control all their basic processes, including
cell cycle, motility, metabolism or genetic expression, to name
just a few.1 Therefore, there is great interest in studying the
role of kinases and phosphatases in natural processes and their
connection with the etiology of diseases such as cancer.2 In this
context, the development of chemical sensors that allow
monitoring of their activity is of great relevance.3 Accordingly,
a number of optical sensors based on environment-sensitive,4
or chelation-enhanced fluorophores5,6 have been described.
Lanthanides, as privileged emissive species,7 have also been
applied to the design of specific kinase sensors, often by taking
advantage of the chelating effect of the phosphate anion to
induce the formation of luminescent lanthanide complexes.8
In the context of our work in the development of fluorescent,9
and lanthanide-based luminescent sensors,10 we envisioned a
new strategy for probing phosphorylation states, and thereby
monitoring the enzymatic activity of kinases and phosphatases,
based on the use of peptide conjugates containing coordinatively-
deficient lanthanide complexes.11 The designed lanthanide-
chelating peptides include a C-terminal Protein Kinase C alpha
(PKCa) recognition sequence,12 a Pro-Gly b-turn inducing
sequence that preorganizes the peptide chain to promote
chelation,13 and a sensing unit consisting of an N-terminal
Peptides were synthesized following standard Fmoc solid-
phase synthesis procedures.18 The Asp and Glu residues were
inserted with their side chains orthogonally protected as allyl
esters, and selectively deprotected by Pd catalysis once the
peptide sequences were fully assembled in the solid phase.19
The DO3A propylamino derivative was then conjugated to
the resulting carboxylic acids.20 The final peptides were
deprotected/cleaved from the resin and purified by reverse-
phase HPLC.
As expected, the peptides were able to coordinate Tb(III)
ions, as shown by the appearance of the typical luminescence
emission bands of the metal (488, 544 and 585 nm) upon
irradiation at 280 nm of equimolar mixtures of the peptide
chelates and TbCl3. Peptides 2[Tb] and 2P[Tb] displayed very
similar spectra with comparable emission intensities, and
therefore these species were not considered for further studies
(Fig. S2, ESIw). Surprisingly, we found that the luminescence
a
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Centro Singular de Investigacion en Quımica Bioloxica e Materiais
´
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Moleculares, Departamento de Quımica Organica and Unidad
Asociada al CSIC. Universidade de Santiago de Compostela,
15782 Santiago de Compostela, Spain.
E-mail: joseluis.mascarenas@usc.es, eugenio.vazquez@usc.es;
Fax: +34 981 505 012; Tel: +34 881 815 738
b Institute of Biochemistry, Faculty of Medicine,
University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.
E-mail: marko.golicnik@mf.uni-lj.si; Fax: +386 15437641;
Tel: +386 1 5437669
w Electronic supplementary information (ESI) available: Peptide
synthesis, luminescence titrations, inner-sphere water calculations,
kinetic studies and data analysis. See DOI: 10.1039/c2cc34958b
Fig. 1 Hypothetical coordination of the lanthanide ion in the alternative
peptide phosphorylation states. The figure shows the sequences of the
Asp, (1[Tb], 1P[Tb]) and Glu-derived (2[Tb], 2P[Tb]) metallopeptides.
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of the unphosphorylated 1[Tb] complex was approximately
2.5 times more intense than that of its phosphorylated counter-
part, 1P[Tb] (Fig. S1 and S3, ESIw). In order to understand
these unexpected relative emission intensities, we measured the
luminescence lifetimes of both species in varying proportions
of H2O/D2O. Fitting the resulting emission decays to single
exponentials provided the metal emission lifetimes for 1P[Tb]
and 1[Tb] in H2O and D2O that were then used to calculate the
number of inner-sphere water molecules bound to the metal
center (q), which turned out to be q E 1 for 1[Tb] and q E 0
for 1P[Tb] (Fig. S4 and S5 and Table S1, ESIw).21 Additionally,
the coordination of the pSer side chain to the lanthanide was
confirmed by 31P NMR; therefore, while 1P, lacking the metal
ion, displayed a clear singlet at 3.63 ppm, complexation with
Eu3+ in 1P[Eu] leads to a complete suppression of the phos-
phorus signal, and no resonance could be observed in the
range of 250 to –250 ppm (Fig. S17, ESIw). In light of these
results it can be concluded that the pSer phosphate is indeed
displacing the inner-sphere water molecule and saturating the
coordination of the lanthanide ion as we intended. The
observed decrease in emission of 1P[Tb] could then be
explained assuming that the coordination of the phosphoserine
side chain in 1P[Tb] pulls the terbium macrocycle away from the
Trp antenna, decreasing the energy transfer, and thus resulting
in weaker emission, even in the absence of the quenching effect
of the displaced water molecule.22,23
Fig. 2 Proposed sensing mechanism by coordination of 1[Eu] with
the external antenna and formation of a luminescent 1[Eu]ÁTNB
complex.
Following on the above results, we reasoned that further
increasing the emission of the unphosphorylated state might
provide an effective phosphatase sensor. Phosphatases have
been linked to perturbations in phosphorylation-mediated
signaling pathways and the occurrence of illnesses, and therefore
the detection of phosphatase activities is of major relevance.24
We envisioned that the desired increase in luminescence might
be achieved by displacing the quenching water molecule with
an external bidentate ligand working as a lanthanide sensitizer.
To test this approach, we selected europium as an emissive ion,
and 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione (TNB) as
external antenna.25 We expected that upon phosphorylation
the intramolecular coordination of the phosphate in the pSer
side chain to the europium ion could displace the antenna,
thereby promoting a decrease in the luminescence emission
(Fig. 2).26
Fig. 3 Left: luminescence titrations of 10 mM solutions of 1[Eu] (K)
and 1P[Eu] (J) monitoring the Eu(III) 5D0
-
7F2 (DJ = 2) hyper-
sensitive transition at 616 nm with increasing concentrations of
TNB,28 and best fit to 1 : 1 and 1 : 2 mixed binding model.29 Right:
luminescence spectra of 10 mM 1[Eu] and 1P[Eu] in the presence of
40 mM TNB. Excitation at 280 nm, 10 mM HEPES, 100 mM NaCl,
pH 7.6. Same scale as the titration.
we decided to explore the use of this sensor for monitoring
the course of enzymatic dephosphorylation (alkaline phos-
phatase, AP), and phosphorylation (PKCa) reactions. Thus,
incubation of optimized concentrations of 1P[Eu] and 1[Eu] in
the presence of TNB with AP or PKCa, respectively, resulted
in the typical response-time profiles shown in Fig. 4.
Irradiation at 280 nm of both peptide complexes in the
presence of TNB resulted in the typical long-wavelength
luminescence emission bands from Eu3+. As expected, the
emission intensity was significantly higher for the unphos-
phorylated peptide 1[Eu] (Fig. 3). Titrations of both peptides
1P[Eu] and 1[Eu] with TNB showed that the formation of the
complex with 1[Eu] was more favorable (KD1 E 11 mM) than
with 1P[Eu] (KD1 E 99 mM).27 Therefore, at appropriate
concentrations, TNB could selectively bind and sensitize the
europium ion in 1[Eu], resulting in a significant enhancement
of the difference in emission between both the unphos-
phorylated and the phosphorylated complexes, so that 1[Eu]
is about 20 times more luminescent than 1P[Eu] in the presence
of the external TNB sensitizer, under the conditions described
in the caption for Fig. 3.
The differential equations governing each reaction step of
the Michaelis–Menten mechanism for dephosphorylation of
1P[Eu] by AP, including the equilibrium accounting for pro-
duct (inorganic phosphate) inhibition, were simultaneously
fitted to the entire set of progress curves (Fig. 4 and Fig. S13,
ESIw). As a result, the Michaelis and inhibition constants were
Km = 14.5 Æ 2.5 mM and Ki = 4.4 Æ 0.6 mM. Likewise, 1[Eu]
was readily phosphorylated by PKCa in the presence of 500 mM
ATP, and the kinase catalysis could also be monitored by the
decrease in the europium emission sensitized by the TNB
antenna, following Michaelis–Menten kinetics with apparent
submicro-molar Michaelis constant Km = 0.23 Æ 0.01 mM
(Fig. 4 and Fig. S16, ESIw).
Having developed Eu(III) peptide complexes that display
large luminescence changes between phosphorylation states,
We have demonstrated that phosphorylation of a serine
residue of designed lanthanide metallopeptides can lead to a
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Chem. Commun., 2012, 48, 9534–9536 9535

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modification of the coordination sphere of the metal ion,
leading to a significant luminescence change. Moreover, we
have shown that addition of an external antenna that displays
selective binding affinity for the non-phosphorylated species
leads to a significant improvement in the ratio between the
emission of the two states of the sensor, allowing the real time
monitoring of enzymatic activity.
We thank the financial support provided by the Spanish grants
SAF2010-20822-C02, CTQ2009-14431/BQU, CSD2007-00006,
Consolider Ingenio 2010, the Xunta de Galicia GRC2010/12,
INCITE09 209 084PR, PGIDIT08CSA-047209PR, Slovenian
Research Agency (grant P1-170 to M.G.). E. P. thanks the
Xunta de Galicia for her postdoctoral contract.
22 Coordination of the Tb to the N-terminal acetyl group, which would
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c
9536 Chem. Commun., 2012, 48, 9534–9536
This journal is The Royal Society of Chemistry 2012