Assessing dimerisation degree and cooperativity in a biomimetic small-molecule model by pulsed EPR

Article abstract of DOI:10.1039/c4cc08656b

Pulsed electron paramagnetic resonance (EPR) spectroscopy is gaining increasing importance as a complementary biophysical technique in structural biology. Here, we describe the synthesis, optimisation, and EPR titration studies of a spin-labelled terpyrid

Full text of DOI:10.1039/c4cc08656b

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Assessing dimerisation degree and cooperativity
in a biomimetic small-molecule model by
pulsed EPR†
Cite this: Chem. Commun., 2015,
1, 5257
5
Received 31st October 2014,
Accepted 8th January 2015
K. Ackermann, A. Giannoulis, D. B. Cordes, A. M. Z. Slawin and B. E. Bode*
DOI: 10.1039/c4cc08656b
www.rsc.org/chemcomm
Pulsed electron paramagnetic resonance (EPR) spectroscopy is the lack of systematic benchmarking using appropriate model
7
gaining increasing importance as a complementary biophysical systems. Here, we demonstrate a tuneable, templated dimerisation
technique in structural biology. Here, we describe the synthesis, in a small-molecule biomimetic model system. We suggest that
optimisation, and EPR titration studies of a spin-labelled terpyridine further studies on homologous synthetic model systems exhibiting
Zn(II) complex serving as a small-molecule model system for tune- different dimerisation equilibria and kinetics could provide the
able dimerisation.
experimental benchmarks required for successful translation of the
PELDOR characteristics involved to corresponding biological
Pulsed (or pulse) electron paramagnetic resonance (EPR) spectro- systems.
scopy can be used to determine reliable distance information on
We have chosen a terpyridine (tpy)-based Zn(II) complex as
1
the nanometre scale. In recent years, the pulsed electron–electron our induced dimerisation model due to the expected high
2
double resonance (PELDOR or DEER) method, in combination binding constant of the ligand to the metal ion facilitating
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with site-directed spin-labelling, has been increasingly employed complex formation. Specifically, the model is built from a
to tackle structural questions on biomacromolecules, such as spin-labelled terpyridine (tpyNO)-based ligand, with Zn(II) as
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proteins and nucleic acids. In addition to the determination of the template. In addition to solvent components, the octa-
inter-spin distances, the local spin concentration can be accessed hedrally coordinated zinc ion binds a maximum of two tridentate
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via the coupling of randomly distributed molecules. Furthermore, ligand molecules, thereby forming the bis-complex Zn(tpyNO)
2
.
the information gained from PELDOR experiments can also be Zn(tpyNO) is the mono-complex and, together with tpyNO,
used to assess the number of spins (‘‘spin counting’’) present in the resembles the monomeric state, while the bis-complex resembles
system under study using the modulation depth (D) of the time the dimer.
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trace. Many proteins form dimers or even higher oligomers in
Metal–tpy complexes have been widely studied in the field of
their biologically active state, a dynamic and often equilibrium- supramolecular chemistry using both dia- and paramagnetic
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1–15
driven process. Inter-monomer contacts observed by X-ray crystallo- metal ions,
and have also been of interest in EPR spectro-
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graphy are not necessarily functionally relevant. Thus, studying scopy for the determination of nitroxide–nitroxide and metal–
dimerisation processes by EPR would be a major advance in our nitroxide distance measurements. While paramagnetic metal
understanding of such systems. However, the number of EPR ions could allow measuring the metal–nitroxide distance,
1
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1
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oligomerisation studies comprising a quantitative assessment of here, we chose the diamagnetic Zn(II) as it does not shorten
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the experimental modulation depth is limited, presumably due to relaxation times of the tpyNO ligands by paramagnetic relaxation
1
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enhancement.
In this proof-of-principle study we monitor metal–tpyNO
complex formation to benchmark the use of PELDOR modulation
depths for quantifying monomer–dimer equilibria. Scheme 1
illustrates the synthesis of the ligand 3 from compounds 1 and
EaStCHEM School of Chemistry, Biomedical Sciences Research Complex and Centre
of Magnetic Resonance, University of St Andrews, North Haugh,
St Andrews KY16 9ST, UK. E-mail: beb2@st-andrews.ac.uk
†
Electronic supplementary information (ESI) available: General experimental
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19,20
information; synthesis and characterisation of compounds; optimisation of the 2. 1 and 2
were obtained by modified literature procedures.
complex and sample preparation for EPR; EPR instrumentation and collection of
Ligand 3 was synthesised by a Sonogashira coupling between 1
and 2. Full details on the syntheses and characterisations are
given in the ESI.†
PELDOR data; additional PELDOR data from the EPR titration series; models for
bis-complex formation; effect of background correction and sample preparation;
determination of dissociation constant and error propagation references. CCDC
The crystal structures of compounds 1 and 3 are shown in
Fig. 1. 1 displays significant disorder in the biphenyl unit. This
1
029722 and 1029723. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c4cc08656b
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Initially, the Zn(II)–tpyNO bis-complex was synthesised and
isolated by precipitation as hexafluorophosphate salt. MALDI
mass spectrometry of this precipitate gave the mass of the
dication confirming the formation of the bis-complex. However,
these samples showed poor solubility and very short phase
memory times. Solubility and phase memory times were signifi-
cantly improved by adding NaBPh
samples for this study were prepared with a ten-fold excess of
NaBPh . All PELDOR distance measurements were performed in
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to the EPR sample. Thus, all
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frozen-solution at 50 K.
For this study, using the in situ generated bis-complex
and NaBPh , the glass-forming solvent system was optimised
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to 80% DMSO-d : 10% D O : 10% ethylene glycol. Deuterated
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2
solvents allow increasing the evolution time of the PELDOR
2
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experiments compared to protonated solvents. Conditions to
obtain the Zn(II)–nitroxide bis-complex were further modified
to allow preparation of a series of small-scale reaction batches
for titration, with each reaction batch containing reagents for
exactly one EPR sample. Comparing PELDOR results from
samples obtained by overnight refluxing with a modified
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‘
‘mix-and-measure’’ procedure showed practically no difference
in modulation depths and overall sample quality. Thus, the
titration series was prepared on a nanomole scale. The putative
structure of the complex used in the titration series is shown
in Fig. 2.
Linearity of the complex is enforced by the octahedral
coordination leading to a trans-configuration of the two central
pyridines bearing the spin-labels. A total of 11 samples was
prepared for the PELDOR titration experiment, and the complete
series was measured 3 times with independent experiments. In
addition, a second titration series was prepared independently to
assess the effect of sample preparation. For both series, the
concentration of the ligand was kept constant, while the Zn(II)/
ligand ratio was varied from 0.0 to 1.0 in 0.1 steps. Thus, the
optimum metal-to-ligand ratio in terms of bis-complex concen-
Scheme 1 Synthesis of the tpy-based ligand (tpyNO).
1
1
tration is expected at 0.5, the point at which each metal ion
is complexed with two ligand molecules. To eliminate the
contribution caused by intermolecular dipolar interactions we
Fig. 1 Crystal structures of compounds 1 (top) and 3 (bottom). Displace-
ment ellipsoids are drawn at 50% probability; hydrogen atoms, solvent
molecules, and minor components of the disorder are omitted for clarity.
is in agreement with the finding that PELDOR experiments on
molecules with similar linkers could only be satisfactorily
simulated by assuming large conformational flexibilities and
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1,22
pronounced bending of the aromatic units.
3 displays
disorder in the biphenyl as 1 does, however, it also shows a
significant deviation from the linearity that might be expected
2
3
from a ‘rigid’ and ‘rod-like’ ligand. This nicely demonstrates
the amount of bending these molecules easily undergo.
Fig. 2 Putative structure of the Zn(II)–tpyNO bis-complex used for the
pulsed EPR titration experiment.
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the samples used in the titration experiment. As expected, the
sample with ratio 0.0 (ligand only) did not show any modulation.
Formation of the bis-complex could be followed by changes in
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the modulation depth (Fig. 3, middle and bottom).
As
expected, a gradual increase in the modulation depth was
observed up to a metal–nitroxide ratio of 0.5. The maximum
modulation depth observed (D Æ root mean square deviation
(
RMSD): 0.325 Æ 0.014) was smaller than expected. The modula-
tion depth of a biradical equals the modulation depth parameter
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(
l) which was found to be 0.43 Æ 0.02 under similar conditions.
However, Fig. 3 shows that all modulation depths seem systema-
tically reduced, suggesting that the stoichiometry of the titration
samples is correct. Furthermore, in our chosen system the
deviation in the expected maximum modulation depth is unlikely
11,15
to be due to incomplete dimerisation.
Potential reasons for
the smaller modulation depth may be linked to the solvent system
used and/or diamagnetic impurities competing with the ligand.
Further increasing the ratio led to a successive decrease in
modulation depth, indicating an increasing concentration of the
mono-complex.
However, even at a ratio of 1 : 1, theoretically allowing 100%
mono-complex, B54% of bis-complex was still present. This
is in good agreement with a previous study, where 60% bis-
complex was found at a 1 : 1 ratio of zinc perchlorate hexa-
1
1
hydrate and tpy, using proton NMR in acetonitrile. To further
investigate the experimental modulation depth, we compared it
to a statistical model (normalised to the experimental modula-
tion depth at ratio 0.5) which does not take into account any
potential cooperativity of the binding of the ligand to the metal
ion, but is purely based on the concentrations present in the
mixture. In theory, assuming a fully positive cooperativity,
modulation depth would not change any more at ratios 40.5
(strong preference for the bis-complex), whereas at fully negative
cooperativity (strong preference for the mono-complex) the modu-
lation depths after ratio 0.5 would mirror the corresponding
values before ratio 0.5. Within the experimental error (given here
Fig. 3 Top: waterfall plot of the background-corrected PELDOR traces. as the RMSD obtained from the DeerAnalysis software, see Fig. 3,
Middle: experimental (ÆRMSD) and modelled modulation depth. Bottom:
respective changes in mean modulation depth in relation to the metal–
nitroxide ratio.
which is in good agreement with the standard deviation obtained
from three independent measurements per sample, see ESI†) the
model closely resembles our data throughout the range of ratios
investigated. This indicates that, in this solvent system and with
exploited the fact that the sample not containing any Zn(II) only our choice of anions, the Zn–tpyNO binding was not cooperative
has intermolecular contributions. This negative control allows (neither positive nor negative; see ESI†). This suggestion is further
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determining the background contribution experimentally.
supported by analysing the actual changes in modulation depth
As the nitroxide concentration and pulse lengths are identical in-between each titration step (Fig. 3, bottom). To assess the effect
for all samples the background contribution (i.e. time constant of sample preparation, a second titration series (i.e. 11 samples
of a monoexponential decay) is known for all samples. Raw data with ratio 0.0 to 1.0) was set up and analysed as before. While
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7
were processed in DeerAnalysis2013. Distance distributions the shape of the titration curve could be accurately reproduced the
were obtained via Tikhonov regularisation using the L-curve actual deviations from the obtained modulation depths were
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criterion.
larger than in-between the independent measurements of the
All derived distance distributions (see ESI†) gave a single first series (see ESI†). This suggests that in most practical cases
well-defined peak with a mean of 5.2(1) nm (the number in the uncertainties introduced during sample preparation and by
parentheses giving the standard deviation, i.e. the peak width background correction will exceed those from reproducibility of
not the error), which is in good agreement with increment modulation depths. It should be noted that the high accuracy
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models.
Fig. 3 shows the PELDOR traces with intermolecular shown in this study owes to the correct determination of the
contributions removed and analysis of the modulation depths of background from the control sample without Zn(II).
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The fraction of dimer present in a monomer/dimer equili-
brium is equal to the ratio D over l. Thus, for a non-templated
dimerisation system such as a protein dimer, the dissociation
constant KD can in principle be derived from the PELDOR
modulation depth and sample concentration (see ESI† for
details).
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We thank the EPSRC National Mass Spectrometry Facility,
Swansea, Mr Stephen Boyer for elemental analyses and
Dr Tomas Lebl and Mrs Melanja Smith for solution NMR
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