Selective protein-surface sensing using Ruthenium(II) Tris(bipyridine) complexes

Article abstract of DOI:10.1002/chem.200902368

"Chemical Equation Presented" Protein surface recognition: Functionalised Ru_II tris-(bipyridine) complexes are shown to act as high-affinity (2 nM) and selective receptors for the surface of cytochrome c (see figure) as compared to a panel of other proteins

Full text of DOI:10.1002/chem.200902368

DOI: 10.1002/chem.200902368
Selective Protein-Surface Sensing Using Ruthenium(II) Tris(bipyridine)
Complexes
James Muldoon,[a, b] Alison E. Ashcroft,[b] and Andrew J. Wilson*[a, b]
Inhibition of protein–protein interactions (PPIs) with de-
signed molecules represents a key challenge in modern bio-
organic chemistry.[1–3] In contrast to competitive enzyme in-
hibition,[4] in which small molecules are optimised to mas-
querade as a substrate (or transition-state analogue) and fit
a well defined concave pocket or cleft, competitive inhibi-
tion of PPIs requires a molecule that must make discontinu-
ous, noncovalent contacts over a much larger (>800 ꢀ2),
surface-lacking, defined shape. Whilst high-throughput
screening has identified inhibitors of some PPIs,[5,6]—these
are generally regarded as ꢁlow-hanging fruitꢂ[2]—there re-
mains a need to develop our basic understanding of how to
design molecules that possess the features needed for pro-
tein-surface recognition. Building on fundamental studies of
short peptide recognition,[7–9] a number of approaches in
which a scaffold is used to project groups capable of making
multivalent hydrophobic,[10] ion-pairing[11–29] and metal–
ligand interactions[30–33] with proteins have been described.
In the current manuscript we illustrate that functionalised
RuII tris-bipyridine complexes can be used as selective and
low nanomolar sensors for cytochrome c (cyt c).[34] Recep-
tors for cyt c have been described[11–13,16–18,20–26] in addition to
inhibitors of its PPIs.[35–37] The current system, however,
offers significant advantages for fundamental studies of pro-
tein-surface recognition. Binding to metalloproteins and
nonmetalloproteins can be detected by using simple fluores-
cence quenching or anisotropy changes, respectively, whilst
structure–affinity studies and screening against a panel of
proteins point to specific interactions with the target. Impor-
tantly, the highest affinity receptor binds cyt c with 1:1 stoi-
chiometry and an affinity of 2 nm.
We selected RuII tris-(5,5’-biscarboxybipyridine) as a core
to which could be appended functional groups capable of
making a diverse array of noncovalent interactions with our
target protein. We then synthesised a series of RuII tris-(5,5’-
biscarboxamidobipyridine) derivatives, 1–5, (as described in
the Supporting Information) which present functional do-
mains of different size and composition that are suitable for
matching to the diverse topology of different proteins.
[a] J. Muldoon, Dr. A. J. Wilson
School of Chemistry
University of Leeds
Woodhouse Lane, Leeds LS2 9JT (UK)
Fax : (+44)113-3436565
E-mail: J.Wilson@leeds.ac.uk
[b] J. Muldoon, Prof. A. E. Ashcroft, Dr. A. J. Wilson
Astbury Centre for Structural Molecular Biology
University of Leeds
Woodhouse Lane, Leeds LS2 9JT (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902368.
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Chem. Eur. J. 2010, 16, 100 – 103

COMMUNICATION
The recognition surface of cyt c centres around a solvent-
exposed hydrophobic haem group surrounded by basic resi-
dues (Figure 1a).[38] As such, we anticipated compounds 2–4,
Figure 2. Fluorescence emission response of receptor 3 (10 nm, 5 mm
sodium phosphate buffer, pH 7.4, lex 467 nm) upon addition of cyt c.
a) Emission spectrum with various concentrations of protein. b) Change
in response at emission maximum of 625 nm and fit by nonlinear regres-
sion (inset: Job Plot). [cyt c] is given in m.
Figure 1. Structures of a) cytochrome c (PDB ID: 1HRC)[39] and its rec-
ognition surface for interaction with other proteins circled in light purple
and b) other proteins tested, including horseradish peroxidase (PDB ID:
1W4W),[40] ferredoxin (PDB ID: 1A7O),[41] myoglobin, (PDB ID:
1HRM),[42] and lysozyme (PDB ID: 2LYM).[43]
which have functional groups capable of cation recognition,
to be potent receptors for this protein. We also selected a
range of additional metallo- and nonmetalloproteins to test
against for selective recognition and sensing (Figure 1b).
These represent proteins of different sizes, and more signifi-
cantly, different surface compositions, but in certain cases
very similar charge (for example, lysozyme and cyt c).
We first tested binding by monitoring the fluorescence re-
sponse of each receptor upon titration with cyt c. A number
of compounds demonstrated almost complete quenching
and saturation behaviour during the experiment. Represen-
tative data are provided for the most potent compound (3)
in Figure 2a and b. The data can be fit to a simple 1:1 bind-
ing isotherm by using nonlinear regression (Figure 2b) to
afford dissociation constants whilst the Job plot (Figure 2b
inset) confirms the expected 1:1 stoichiometry. Shown in
Figure 3 are the titration curves for binding of cyt c to each
of the metal complexes. Unsurprisingly, the charge-mis-
matched, amino-functionalised receptor 5 does not bind to
the target protein, whilst those compounds with increasing
numbers of aspartate residues exhibit progressively stronger
binding. Compound 3 binds with a dissociation constant of
Figure 3. Change in fluorescence emission response of receptors 1–5 at
lem maximum of 625 nm upon titration with cyt c (conditions as for
Figure 3). [cyt c] is given in m.
2 nm, which is amongst the strongest affinities yet observed
for the binding of cyt c by a synthetic receptor[16] and repre-
sents five orders of magnitude increase in affinity over the
core scaffold. This is very dramatic considering that other
scaffolds (for example, porphyrins) exhibit significant affini-
ty for the protein,[11,13] whereas the majority of affinity in
our case derives from functionalisation. We attempted to
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A. J. Wilson et al.
Table 1. Dissociation constants for binding of receptor 3 to different pro-
evaluate the role of multivalency by increasing the number
of carboxylates going from compounds 1–3. The average
binding free energy per carboxylate is À4.68 kJmolÀ1 for 1,
À3.66 kJmolÀ1 for 2 and À2.08 kJmolÀ1 and for 3; this indi-
cates negative co-operativity. Negatively co-operative bind-
ing interactions are commonly observed.[44] This is not unex-
pected, however, given that our current analysis is somewhat
crude in that other noncovalent contacts (for example, hy-
drophobic) are likely to be important and only a certain
number of carboxylates will make contacts with the pro-
tein—a phenomenon observed in dendrimer–DNA interac-
tions.[45] We were surprised to find that tris-(5,5’-biscarboxa-
midobipyridine) (4), which is functionalised with a crown
motif, exhibited virtually no binding to cyt c. We had antici-
pated that the crown ring might recognise lysine residues in
preference to arginine residues and thus prove useful for dif-
ferentiating between basic proteins with different side
chains. One reason for this might be that the multivalent
contacts that could be made upon cyt c binding are not out-
weighed by the preferred binding to excess cations present
in buffer. Pleasingly, the individual ligands do not appear to
bind with high affinity to cyt c. Due to the absence of diag-
nostic spectroscopic properties, compound 6 (which repre-
sents the ligand comprising 3) was tested in a competition
assay against the cyt c--1 interaction. Unfortunately, recov-
ery of the fluorescence of 1 upon displacement from the sur-
face of cyt c was not observed—only minor changes in fluo-
rescence at high mm concentrations were seen (see the Sup-
porting Information, Figure 6). This suggests weak binding,
however it is not conclusive as there are other interpreta-
tions of this result (for example, both species could be bind-
ing to different sites).
Following these initial binding studies we tested each of
the compounds in a functional assay. The reduction kinetics
of cyt c by ascorbate have previously been shown to be
modified by the binding of receptors to its haem-exposed
edge.[12,46,47] Specifically, a retarded rate of reduction is pro-
posed to arise as a result of binding and steric blockage of
the reducing agent from approach to the haem group. As
expected, at a single concentration, the receptors retarded
the rate of reduction in line with their binding affinity deter-
mined by fluorescence (see the Supporting Information)
that is, receptor 3 exhibited potent inhibition of reduction
whilst receptor 5 exhibited no change. These results are
therefore indicative of binding to the surface of cyt c as pro-
posed. Furthermore, we were also able to observe the inter-
action of cyt c and the receptor by using mass spectrometry.
ESI mass spectra (see the Supporting Information) of the
protein in the presence of receptor 3 clearly showed the
presence of noncovalently bound 1:1 and 2:1 products (the
observation of two molecules of 3 bound to cyt c in the mass
spectrum is not unsurprising given that ESI enhances elec-
trostatic interactions). This technique could be used for
rapid screening of this class of receptors against a panel of
proteins in future.
teins[a]
Protein
Dissociation constant (Kd) [nm]
cytochrome c
acetylated cytochrome c
myoglobin
ferredoxin
horseradish peroxidase
lysozyme
2Æ0.5
>30000
>30000
>30000
>30000
273Æ43
[a] Conditions for titration as indicated in Figure 2.
tested no fluorescence response was observed (Table 1).
Whilst a noncovalent interaction cannot be excluded on the
basis of the data, this seems likely and the results clearly
demonstrate that selective sensing of proteins is possible.
Most significantly, acetylated cyt c in which the lysines have
been blocked did not respond to receptor 3; this suggests
again that molecular recognition derives from interaction of
receptor 3 and the basic surface of cyt c. We were also eager
to study binding with nonmetalloproteins. Binding, however,
does not necessarily result in changes to the fluorescence
signal. We therefore monitored changes in both fluorescence
intensity and fluorescence anisotropy (in which the signal is
dependent upon the molecular weight of the species in solu-
tion). Receptor 3 experienced a dose-dependent change in
anisotropy signal upon titration with lysozyme, but no
changes in emission intensity. The data could be fit to a 1:1
model with a dissociation constant of 273 nm. What is most
dramatic about this result is that lysozyme and cyt c have a
similar charge state (i.e. pI) and surface composition, yet re-
ceptor 3 exhibits two orders of magnitude higher affinity for
the latter protein pointing to a structurally well-defined in-
teraction that extends beyond a simple ꢁgrease and chargeꢂ
model of interaction!
In conclusion, we have illustrated that functionalised RuII
tris-bipyridine complexes act as selective and high affinity
receptors for proteins surfaces. These metal complexes
could similarly form the basis of protein sensors for diagnos-
tic purposes.[48–52] Our future studies will focus upon under-
standing the binding interaction within this model system at
a structural and thermodynamic level and inhibiting phar-
maceutically relevant PPIs.
Acknowledgements
This work was supported by the Wellcome Trust [WT 78112/Z/05/Z].
Keywords: cytochrome c
· protein–protein interactions ·
protein–surface recognition · supramolecular chemistry
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Received: August 26, 2009
Published online: November 27, 2009
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