Directed Self-Assembly of Trimeric DNA-Bindingchiral Miniprotein Helicates

Abstract

We propose that peptides are highly versatile platforms for the precise design of supramolecular metal architectures, and particularly, for the controlled assembly of helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff) can been engineered on its N-terminus with metal-chelating 2,2′-bipyridine units that stereoselectively assemble in the presence of Fe(II) into parallel, three-stranded peptide helicates with preferred helical orientation. Modeling studies support the proposed self-assembly and the stability of the final helicate. Furthermore, we show that these designed mini-metalloproteins selectively recognize three-way DNA junctions over double-stranded DNA.

Full text

ORIGINAL RESEARCH
published: 30 October 2018
doi: 10.3389/fchem.2018.00520
Frontiers in Chemistry | www.frontiersin.org 1October 2018 | Volume 6 | Article 520
Edited by:
Angela Casini,
Cardiff University, United Kingdom
Reviewed by:
Olga Iranzo,
Center National de la Recherche
Scientifique Marseille, France
Guzman Gil-Ramirez,
University of Lincoln, United Kingdom
*Correspondence:
Jean-Didier Maréchal
jeandidier.marechal@uab.cat
Miguel Vázquez López
miguel.vazquez.lopez@usc.es
M. Eugenio Vázquez
eugenio.vazquez@usc.es
Specialty section:
This article was submitted to
Supramolecular Chemistry,
a section of the journal
Frontiers in Chemistry
Received: 20 August 2018
Accepted: 09 October 2018
Published: 30 October 2018
Citation:
Gómez-González J, Peña DG,
Barka G, Sciortino G, Maréchal J-D,
Vázquez López M and Vázquez ME
(2018) Directed Self-Assembly of
Trimeric DNA-Bindingchiral Miniprotein
Helicates. Front. Chem. 6:520.
doi: 10.3389/fchem.2018.00520
Directed Self-Assembly of Trimeric
DNA-Bindingchiral Miniprotein
Helicates
Jacobo Gómez-González 1, Diego G. Peña 2, Ghofrane Barka 1, Giuseppe Sciortino 3,4 ,
Jean-Didier Maréchal 3
*, Miguel Vázquez López 1
*and M. Eugenio Vázquez 2
*
1Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química
Inorgánica, Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 2Centro Singular de Investigación en
Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Orgánica, Universidade de Santiago de
Compostela, Santiago de Compostela, Spain, 3Departament de Química, Universitat Autònoma de Barcelona, Cerdanyola,
Spain, 4Dipartimento di Chimica e Farmacia, Università di Sassari, Sassari, Italy
We propose that peptides are highly versatile platforms for the precise design of
supramolecular metal architectures, and particularly, for the controlled assembly of
helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff)
can been engineered on its N-terminus with metal-chelating 2,2′-bipyridine units that
stereoselectively assemble in the presence of Fe(II) into parallel, three-stranded peptide
helicates with preferred helical orientation. Modeling studies support the proposed
self-assembly and the stability of the final helicate. Furthermore, we show that these
designed mini-metalloproteins selectively recognize three-way DNA junctions over
double-stranded DNA.
Keywords: metallopeptide, self-assembly water, DNA recognition, enantioselectivity, peptide motifs, coordination
chemistry
INTRODUCTION
Peptides are excellent supramolecular building blocks that encode precise structural and functional
information within their amino acid sequence. Accordingly, researchers have explored diverse
peptide motifs, such as coiled-coils, β-hairpins, or peptide amphiphiles, as the basis of biofunctional
devices and materials (Matsuura et al., 2005, 2010; Gazit, 2007; Ulijn and Smith, 2008; Apostolovic
et al., 2010; Robson Marsden and Kros, 2010; Boyle and Woolfson, 2011; Lai et al., 2012;
Pazos et al., 2016). Curiously, despite the enormous potential for controlling stereochemistry,
nuclearity and stoichiometry, the controlled supramolecular assembly of inorganic complexes
with peptide motifs has been somewhat overlooked, and only a handful of systems based on
modified coiled-coil motifs have been reported (Lieberman and Sasaki, 1991; Ghadiri et al.,
1992; Li et al., 2000; Peacock et al., 2012; Ball, 2013; Berwick et al., 2014; Luo et al., 2016).
On the other hand, helicates are discrete metal complexes in which one or more organic
ligands are coiled around—and coordinating—two or more metal ions (Piguet et al., 1997;
Albrecht, 2001, 2005) as a result of ligand coiling, helicates are inherently chiral species
that can appear as two enantiomers according to the orientation in which the ligands twist
around the helical axis defined by the metal centers. Besides their intrinsic interest in basic
supramolecular chemistry, helicates have shown promising DNA-binding properties that have
been associated with antimicrobial and antitumoral effects (Howson et al., 2012; Kaner et al.,
2015). However, more than 20 years after the pioneering studies by Prof. Jean-Marie Lehn
(Lehn et al., 1987; Ulijn and Smith, 2008), helicates are still not viable alternatives to traditional

Gómez-González et al. Miniprotein Helicates
DNA-binding agents. The slow development in the applied
chemistry of metal helicates ultimately derives from the
shortcomings associated with the classic synthetic approaches
with organic ligands that complicate the structural control of the
final helicates (i.e., oligomerization state, relative orientation of
asymmetric ligands, supramolecular helicity) and hampers their
efficient structural and functional optimization. Indeed, despite
some noteworthy examples (Haino et al., 2009; Cardo et al., 2011;
Howson et al., 2012; Chen et al., 2017; Mitchell et al., 2017; Guan
et al., 2018), no general approach for the efficient and versatile
stereoselective synthesis of helicates is yet available, making of
these systems a challenging test case to demonstrate the potential
of peptides for the controlled assembly of metallostructures.
Our strategy relied in the selection of a synthetically-accessible
and structurally well-defined trimeric peptide domain as scaffold
for the programmed assembly of the helicate. As an alternative
(and orthogonal) platform to the ubiquitous leucine zippers,
we focused our attention on the C-terminal domain of the
bacteriophage T4 Fibritin foldon (T4Ff), a trimeric β-propeller-
like structure formed by the self-assembly of a short 27-amino
acid peptide (Tao et al., 1997; Papanikolopoulou et al., 2004;
Habazettl et al., 2009). The intrinsic stability and structural
resilience of the T4Ff scaffold has been exploited for the
stabilization of trimeric structures of a number of peptides
and engineered proteins (Stetefeld et al., 2003; Du et al., 2011;
Berthelmann et al., 2014; Kobayashi et al., 2015), and given
those precedents we envisioned that the T4Ff could also be used
as a robust platform for the programmed assembly of chiral
dinuclear helicates, thus offering an alternative for the integration
of coordination and peptide chemistry beyond other widely
explored peptide scaffolds.
MATERIALS AND METHODS
General
All reagents were acquired from the regular chemical suppliers.
All solvents were dry and synthesis grade, unless specifically
noted (NH4)Fe2(SO4)2•6 H2O salt from Sigma-Aldrich was
used as Fe(II) ion source. Reactions were followed by analytical
UHPLC-MS with an Agilent 1200 series LC/MS using a SB C18
(1.8 µm, 2.1 ×50 mm) analytical column from Phenomenex.
Standard conditions for analytical UHPLC consisted on a linear
gradient from 5 to 95% of solvent B for 12 min at a flow rate
of 0.35 mL/min (A: water with 0.1% TFA, B: acetonitrile with
0.1% TFA). Compounds were detected by UV absorption at 222,
270, and 330 nm. Electrospray Ionization Mass Spectrometry
(ESI/MS) was performed with an Agilent 6120 Quadrupole
LC/MS model in positive scan mode using direct injection of the
purified peptide solution into the MS detector.
Computational Methods
The model for the 33-[(βAlaBpy)2-T4Ff]3Fe+4
2helicate was
built with UCSF chimera1.12 (Pettersen et al., 2004), starting
from the NMR resolved structure of the trimeric Foldon
of the T4 phagehead fibritin (PDB code: 1RFO) mutating
the carboxyl C-Termini to amide groups (see Results and
Discussion section). Based on previous work, the model of
33-[(βAlaBpy)2-T4Ff]3Fe+4
2helicate were built connecting the
N-termini of the T4Ff peptides. Molecular Dynamics (MD)
simulations were set up with the xleap, solvating the model
with a box of pre-equilibrated TIP3P water molecules and the
total charge was balanced with Cl−ions (ions94.lib library). The
AMBER14SB force field was used for standard residues (Hornak,
Abel, Okur, Strockbine, Roitberg and Simmerling., 2006), while
the GAFF force field was adopted for the remaining atoms.
Fe-bonding force constants and equilibrium parameters were
obtained through the Seminario method, using Gaussian09 to
compute the geometry and harmonic frequencies at DFT level
(Frisch et al., 2010), with the B3LYP functional (Yanai et al.,
2004), combined with scalar-relativistic Stuttgart–Dresden SDD
pseudopotential and its associated double-ζbasis plus a set of
fpolarization functions for the metal ion (Ehlers et al., 1993).
The 6-31G(d,p) basis set was used for H, C, O, and N. Point
charges were derived using the RESP (Restrained ElectroStatic
Potential) model (Bayly et al., 1993). The force field building
operations were carried out using the MCPB.py (Li and Merz,
2016). The solvent and the whole system were sequentially
submitted to 3,000 energy minimization steps to relax possible
steric clashes. Then, thermalization of water molecules and side
chains was achieved by increasing the temperature from 100 K up
to 300 K. MD simulations under periodic boundary conditions
were carried out during 100 ns with OpenMM engine through
OMMProtocol (Eastman et al., 2017; Pedregal et al., 2018).
Analysis of the trajectories was carried out by means of cpptraj
implemented in ambertools16 (Case et al., 2016).
Solid-Phase Peptide Synthesis (SPPS)
All peptide synthesis reagents, as well as the Fmoc amino acid
derivatives were purchased from GL Biochem (Shanghai) Ltd.,
Fmoc-β-Ala-OH was from Sigma Aldrich. C-terminal amide
natural T4Ff peptides were synthesized following standard Fmoc-
peptide synthesis protocols on a 0.1 mmol scale using a 0.5
mmol/g loading H-Rink amide ChemMatrix resin (35–100 mesh)
from Sigma Aldrich with a Liberty Lite automatic microwave
assisted peptide synthesizer from CEM Corporation. The amino
acids were coupled in 5-fold excess using oxyme as an activating
agent. Couplings were conducted for 4 min at 90◦C. Deprotection
of the temporal Fmoc protecting group was performed by
treating the resin with 20% piperidine in DMF for 1 min at
75◦C. Once the synthesis is finished, the peptide was acetylated
with a solution of 0.8 ml AcOH, 2 ml of DIEA/DMF (0.2 M)
and 3.2 ml of DMF. The last non-natural Fmoc-β-Ala-Bpy-OH
residues were coupled by hand in 4-fold excess using HATU
as activating agent. Each amino acid was activated for 1 min
in DIEA/DMF 0.2 M before being added onto the resin. These
manual couplings were conducted for 60 min. Deprotection of
the temporal Fmoc protecting group was performed by treating
the resin with 20% piperidine in DMF for 20 min. Cleavage and
deprotection of the peptide were simultaneously performed using
standard conditions by incubating the resin for 2.5 h with an
acidic mixture containing 50 µL CH2Cl2, 25 µL of H2O, 25
µL of TIS (triisopropylsilane), and 900 TFA µL. The resin was
filtered, and the TFA filtrate was concentrated under a nitrogen
stream to an approximate volume of 1 mL, and then added onto
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Gómez-González et al. Miniprotein Helicates
ice-cold diethyl ether (20 mL). After 10–30 min, the precipitate
was centrifuged and washed again with 5 mL of ice-cold ether.
The solid residue was dried under argon and redissolved in
acetonitrile/water 1:1 (2–5 mL) and purified by semi-preparative
RP-HPLC.
Peptides were purified by preparative RP-HPLC with an
Waters 1500 series Liquid Chromatograph using a Sunfire
Prep C18 OBD (5 µm, 19 ×150 mm) reverse-phase column
from Waters. Standard conditions for analytical and preparative
RP- HPLC consisted on an isocratic regime during the first
2 min, followed by a linear gradient from 15 to 75% of
solvent B for 30 min (A: water 0.1% TFA, B: acetonitrile 0.1%
TFA). Compounds were detected by UV absorption (222 nm)
and by ESI/MS. The fractions containing the products were
freeze-dried and their identity was confirmed by ESI/MS
and MALDI-TOF. Matrix-assisted laser desorption/ionization
mass spectrometry (MALDI/MS) was performed with a Bruker
Autoflex MALDI/TOF model in positive scan mode by direct
irradiation of the matrix-absorbed peptide.
Spectroscopic Measurements
UV measurements were made in a Jasco V-630
spectrophotometer coupled to a Jasco ETC-717 temperature
controller, using a standard Hellma semi-micro cuvette (108.002-
QS) with a light path of 10 mm. Measurements were made at
20◦C. Luminescence experiments were made with a Varian
Cary Eclipse Fluorescence Spectophotometer coupled to a Cary
Single Cell peltier accessory (Agilent Technologies) temperature
controller. All measurements were made with a Hellma
semi-micro cuvette (108F-QS) at 20◦C. Circular dichroism
measurements were made with a Jasco J-715 coupled to a Neslab
RTE-111 termostated water bath, using a Hellma 100-QS cuvette
(2 mm light pass).
Electrophoretic Mobility Shift Assays
EMSA were performed with a BioRad Mini Protean gel system,
powered by an electrophoresis power supplies PowerPac Basic
model, maximum power 150 V, frequency 50–60 Hz at 140 V
(constant V). Binding reactions were performed over 30 min in
1.8 mM Tris-HCl (pH 7.5), 90 mM KCl, 1.8 mM MgCl2, 0.2 mM
TCEP, 9% glycerol, 0.11 mg/mL BSA, and 2.2% NP-40. For the
experiments we used 200 nM of the DNAs (twDNA and dsDNA),
and a total incubation volume of 20 µL. After incubation for
30 min at room temperature, products were resolved by PAGE
using a 10% non-denaturing polyacrylamide gel and 1 ×TBE
buffer (0.445 M Tris, 0.445M Boric acid) for 35 min at 25◦C, and
analyzed by staining with SyBrGold (Molecular Probes: 5 µL in
50 mL of 0.5 ×TBE) for 10 min and visualized by fluorescence
(BioRad GelDoc XR+molecular imager).
RESULTS AND DISCUSSION
As metal-chelating unit we chose 2,2′-bipyridine, a ligand that
has been extensively used in coordination chemistry and yields
stable complexes with a variety of metal ions (Kaes et al.,
2000). Furthermore, we have previously described an Fmoc-
protected 2,2′-bipyridine dipeptide derivative that can be readily
implemented into standard Fmoc solid-phase peptide synthesis
(SPPS) protocols, and have showed that the structure of this
chelating unit, in which the 2,2′-bipyridine ligand is integrated
in the peptide backbone, effectively couples the conformational
preferences of the peptide chain with the geometry of the
resulting metal complexes (Rama et al., 2012; Gamba et al., 2013,
2014, 2016; Salvadó et al., 2016).
The chelating 2,2′-bipyridine residue was obtained following
an optimized synthetic route (Rama et al., 2012), based
on the work carried out by the Newkome and Imperiali
groups (Newkome et al., 1997; Torrado et al., 1998). The
key step in the synthesis being the desymmetrization of
a diethyl [1,1′-biphenyl]-4,4′-dicarboxylate intermediate with
hydrazine monohydrate under conditions that allow the selective
precipitation of the monocarbohydrazide, which is oxidized
into the corresponding azyl azide, and then transformed into a
carbamate through a Curtius rearrangement (Rama et al., 2012).
Simultaneous hydrolysis of the carbamate and the ester group
gives the desired bipyridine amino acid, which is derivatized in
the form of a dipeptide to obtain the Fmoc-βAlaBpy-OH building
block for increased solubility, stability, and solubility that allow
its use following standard solid-phase peptide synthesis protocols
(Ishida et al., 2006).
Inspection of the structure of T4Ff (PDB IDs 4NCU or
1RFO; Güthe et al., 2004; Berthelmann et al., 2014) showed
that the N-terminal Gly residues are relatively close to each
other and could accommodate the chelating 2,2′-bipyridine units
without noticeable distortion of the T4Ff scaffold upon metal
coordination. Moreover, we envisioned that the natural twist
of the N-terminal polyproline helices in the folded T4Ff trimer
should induce a 33-configuration (Mhelicity) on its derived
helicate (Tao et al., 1997), which would be the preferred chirality
for the efficient recognition of three-way DNA junctions (Oleksy
et al., 2006; Gamba et al., 2016). Therefore, we synthesized
the desired (βAlaBpy)2-T4Ff helicate precursor ligand following
standard Fmoc SPPS methods as outlined in Figure 1 (Coin et al.,
2007). The final peptide ligand was purified by HPLC and its
identity confirmed by ESI-MS.
Having at hand the desired peptides we proceeded with
the study of their metal binding properties. Surprisingly, while
2,2′-bipyridine is weakly emissive, and is even considered
non-fluorescent (Dhanya and Bhattacharyya, 1992; Yagi et al.,
1994), we found that the asymmetric 5′-amido-[2,2′-bipyridine]-
5-carboxamide unit within the βAlaBpy residue was highly
emissive, displaying intense band at c.a. 420 nm with a quantum
yield of 0.37 (Dong et al., 2017). Additionally, the emission
was quenched by coordination to Fe(II) ions, which could be
exploited to monitor the formation of the β-annulus helicate.
Thus, we recorded the emission spectra of a 3 µM solution
(9 µM monomer) of [(βAlaBpy)2-T4Ff]3in phosphate buffer
(1 mM, pH 6.5) in the presence of increasing concentrations
of (NH4)2Fe(SO4)2•6 H2O (Mohr salt) as source of Fe(II)
ions (λexc =305 nm), and obser ved a concentration-dependent
quenching of the emission intensity of the bipyridine ligands.
The emission intensity profile of the titration nm could be fitted
to a 1:2 binding mode with dissociation constants for the first,
and second iron coordination of KD1=5.5 ±3.3 µM and a
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Gómez-González et al. Miniprotein Helicates
FIGURE 1 | (A) Structural elements and sequence of the natural T4Ff, and proposed structure of the (βAlaBpy)2-T4Ff helicate at the N-terminus of the T4Ff scaffold.
The three chains of the T4Ff are shown with different colors (orange, blue, light gray) for clarity. The 33- chirality is induced by the natural twisting of the T4Ff
N-terminal polyproline helices. (B) Synthetic procedure for obtaining the T4Ff helicates, and structure of the chelating Fmoc-βAlaBpy-OH amino acid.
KD2=6.6 ±0.7 µM, respectively (Figure 2, left; Kuzmic, 1996,
2009). UV/Vis titrations were also qualitatively consistent with
the fluorescence data, showing a weak MLCT at about 535 nm in
the presence of Fe(II) ions (See Supplementary Material). The
formation of the expected [[(βAlaBpy)2-T4Ff]3Fe2]4+complex
was also confirmed by mass spectrometry of the final solution of
the titrations, which showed a peak at the expected mass of the
molecular ion (m/z=11084.6).
In order to study the chirality induction around the
metal centers we measured the circular dichroism spectra
of the trimeric [(βAlaBpy)2-T4Ff]3ligand, and its Fe(II)
complex [(βAlaBpy)2-T4Ff]3Fe+4
2. As expected from the original
structural analysis, the observed positive Cotton effect at c.a.
330 nm is consistent with the formation of a 33-helicate.
Furthermore, the small change in the CD spectra upon
addition of Fe(II) also suggests that the bipyridine ligands
are strongly preorganized, even in absence of the metal, and
that only a small rearrangement of the chromophores takes
place upon coordination (Figure 2, right). This is consistent
with earlier computational studies with related bis-bipyridyl
peptide ligands, which showed that the bipyridine residues have
a large tendency to stack on top of each other (Rama et al.,
2012). This stacking interaction will presumably rigidify the bis-
bipyridyl trimer and facilitate the helical induction by the foldon
domain.
In order to gain some insight into the structure and stability
of the peptide helicate we performed Molecular Dynamics (MD)
simulations in explicit solvent and periodic boundary conditions
(see Methods section for details). The structure of the 33-
[(βAlaBpy)2-T4Ff]3Fe+4
2unit appears highly stable along all the
MD trajectory retaining its helicity conformation and the Fe(II)
octahedral coordination geometry. Moreover, the T4Ff scaffold
appears stable during the simulation showing no appreciable
deformations as a result of the introduction of the artifical
(βAlaBpy)2unit. The root-mean square deviation (RMSD) of the
whole system was computed along the MD using the minimized
initial structures as a reference, the trajectories attain relative
stable RMSD after the first ∼20 ns, that reach up to 1.99 ±0.62
Å in average (See Supplementary Material). A cluster analysis
was performed on the full length MD experiments showing a
predominant conformations occupying about ∼40% of the total
conformation repartition. Overall, the results highlight that the
computed model is very stable along the 100 ns of the MD and
results consistent with the experimental data. Interestingly, the
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Gómez-González et al. Miniprotein Helicates
FIGURE 2 | Fluorescence titration of a 3 µM (9 µM monomer) solution of
[(βAlaBpy)2-T4Ff]3with increasing concentrations of Fe(II). Inset shows
emission at 420 nm upon excitation at 305 nm with increasing concentrations
of Fe(II), and the best fit to a 1:2 binding mode (Hellman and Fried, 2007;
Peberdy et al., 2007). Experiments were made in triplicate. Right. Circular
Dichroism of a 6 µM solution (18 µM monomer) of [(βAlaBpy)2-T4Ff]3
(dashed line) and in the presence of 90 µM Fe(II) (solid line). All experiments
were made in 1 mM phosphate buffer, pH 6.5, 10 mM NaCl at 20◦C.
FIGURE 3 | First cluster representative frame of the MD trajectory for the
33-[(βAlaBpy)2-T4Ff]3Fe+4
2system showing the stable structure of the T4Ff
domain. Note the flexible hinge region between the rigid helicate and the T4Ff
domain.
MD analysis revealed a hinge region with increased flexibility
connecting the more rigid helicate and foldon domains, which
suggests the replacement of the N-terminal Gly reside for a more
conformationally restricted residue in future designs.
Having made a preliminary characterization of the T4Ff
helicate, we studied its DNA binding properties by titrating
a 2 µM solution of [(βAlaBpy)2-T4Ff]3(6 µM mononer) in
the presence of saturating concentrations of Fe(II) according
to the previous fluorescence titrations (20 µM) with increasing
concentrations of a three-way DNA junction (tw-DNA),
and measuring the fluorescence anisotropy of the bipyridine
fluorophores at 420 nm after each addition of DNA. The
titration profile could be fitted to a 1:1 binding mode, with a
FIGURE 4 | (Left) Anisotropy titration of [(βAlaBpy)2-T4Ff]3Fe2in 1 mM
phosphate buffer, 10 mM NaCl with increasing concentrations of tw-DNA. The
best fit to a 1:1 binding mode is shown (curve fitting was performed using
DynaFit).(Kuzmic, 1996, 2009)tw-DNA sequences: 5′–CAC CGC TCT GGT
CCT C−3′; 5′–CAG GCT GTG AGC GGT G−3′; 5′–GAG GAC CAA CAG CCT
G−3′. Right: Model of the interaction between the [(βAlaBpy)2-T4Ff]3Fe2
and the three-way junction, based on the reported pdb structures of an
helicate bound to a three-way junction (pdb code 4NCU), and the structure of
the fibritin foldon (pdb code 2ET0; Oleksy et al., 2006).
dissociation constant of 2.17 ±0.45 µM of the [(βAlaBpy)2-
T4Ff]3Fe2complex to tw-DNA. Titrations under the same
conditions with a model double stranded DNA (ds-DNA) led
to a small, monotonic increase in the anisotropy, which is in
tune with the the formation of weak complexes or non-specific
binding (Figure 3). The low affinity to dsDNA is consistent
with previous studies with other helicates (Figure 4;Tuma
et al., 1999; Oleksy et al., 2006; Gamba et al., 2016). Control
titrations adding with [(βAlaBpy)2-T4Ff]3foldon in absence
of metal did not show any response to added DNA (See
Supplementary Material), thus confirming that the formation
of the helicate structure is required for DNA recognition, and
the foldon only have a structural role in the formation of the
helicate.
In addition to the spectroscopic studies, we also studied the
DNA binding properties of the [(βAlaBpy)2-T4Ff]3Fe2helicate
by electrophoretic mobility assays (EMSA) in polyacrylamide
gel under non-denaturing conditions (Liebler and Diederichsen,
2004), visualizing the DNA in the gel using SybrGold staining
(Vázquez et al., 2007). In agreement with the fluorescence
titration studies discussed previously, incubation of the target
tw-DNA with the [(βAlaBpy)2-T4Ff]3Fe2helicate resulted in
the concentration-dependent appearance of a new retarded
band, which is consistent with the formation of the expected
tw-DNA/[(βAlaBpy)2-T4Ff]3Fe2complex (Figure 5, lanes 1–
6). Additionally, the overall intensity of the lanes of the
gel is progressively reduced in the presence of increasing
concentrations of the [(βAlaBpy)2-T4Ff]3Fe2complex, which
suggests the formation of higher-order aggregates with the three-
way junction DNA in the gel conditions (Chanvorachote et al.,
2009; Thordarson, 2010). On the other hand, incubation of
a model double-stranded DNA with the peptide helicate did
not show any new slow-migrating bands (Figure 5, lanes 7-10),
which is in agreement with the expected low affinity for this
form of DNA, and demonstrates that the small increase observed
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Gómez-González et al. Miniprotein Helicates
FIGURE 5 | EMSA DNA binding studies results for [(βAlaBpy)2-T4Ff]3Fe2
helicate. Lanes 1–6, 200 nM tw-Rho-DNA with 0, 150, 250, 500, 1,000, and
2,000 nM of [(βAlaBpy)2-T4Ff]3and 14 eq. of (NH4)2Fe(SO4)2•6 H2O in
each lane; lanes 7–10, 200 nM dsDNA with 0, 500, 1,000, and 2,000 nM of
[(βAlaBpy)2-T4Ff]3and 14 eq. of (NH4)2Fe(SO4)2•6 H2O in each lane.
Samples were resolved on a 10% nondenaturing polyacrylamide gel and 1×
TBE buffer over 35 min at 25◦C, and stained with SyBrGold (5 µL in 50 mL of
0.5×TBE) for 10 min, followed by fluorescence visualization. Oligonucleotide
sequences: tw-DNA, 5′–CAC CGC TCT GGT CCT C−3′; 5′–CAG GCT GTG
AGC GGT G−3′; 5′–GAG GAC CAA CAG CCT G−3′;dsDNA (only one
strand shown) 5′–AAC ACA TGC AGG ACG GCG CTT−3′.
in the fluorescence anisotropy titration of dsDNA (Figure 4)
arises from weak interactions that are not seen at the lower
concentrations used in the EMSA experiment.
CONCLUSIONS
In summary, we have shown the potential of small protein
domains for the precise structural organization of coordination
complexes. Modification of the T4 Fibritin foldon with metal-
chelating bipyridines results allows the assembly of unique
three-strand helicates in which the parallel orientation of the
three helicate ligands is directed by the self-assembled T4Ff
domain, and the chirality of the dinuclear helicate (Mhelicity
or 33-configuration in the metal complexes) is selected by the
relative orientation of the natural polyproline helices at the N-
terminus of the T4Ff trimer. The final supramolecular peptide
helicate [(βAlaBpy)2-T4Ff]3Fe2displays good in vitro DNA
binding and selectivity toward three-way DNA junctions. We
are currently exploring alternative peptide sequences to improve
the solubility of the peptide/DNA complexes, and modifications
with positively charged residues that might increase the overall
affinity.
AUTHOR CONTRIBUTIONS
JG-G and DGP performed the experimental work (synthesis
of the bipyridine building block, peptide synthesis, metal
and DNA binding studies), GB did preliminary studies
with the (βAlaBpy)2-T4Ff peptide. GS and J-DM did the
computational work and contributed to the preparation of
the final manuscript. MVL and MEV conceived the project,
supervised the experimental work. MEV wrote the manuscript
with the collaboration of MVL, and prepared the graphic
material.
ACKNOWLEDGMENTS
Financial support from the Spanish grants CTQ2015-70698-R,
CTQ2017-87889-P, the Xunta de Galicia (Centro singular de
investigación de Galicia accreditation 2016–2019, ED431G/09)
and the European Union (European Regional Development
Fund - ERDF), is gratefully acknowledged. JG-G, thanks the
Spanish MINECO for his FPI fellowship, GB thanks the ERC
for her EU METALIC-II 2013-2442/001-001-EMA2 mobility
scheme fellowship, and GS. thanks the Universitat Autònoma
de Barcelona for its support to his PhD. J-DM and GS are
thankful for the support given by the Generalitat de Catalunya
2017SGR1323. Support of COST Action CM1306 is kindly
acknowledged. MEV, also wish to acknowledge the generous
support by the Fundación Asociación Española Contra el Cáncer
AECC (IDEAS197VAZQ grant).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fchem.
2018.00520/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
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