Metal-triggered radial self-assembly of collagen peptide fibers

Article abstract of DOI:10.1021/ja804942w

A metal-triggered self-assembling collagen peptide was designed and synthesized to generate fibers through a radial growth mechanism. The assembly of the fibers was made possible through the placement of a bipyridine ligands within the center of the triple helix and was triggered by the addition of Fe(II). Copyright

Full text of DOI:10.1021/ja804942w

Published on Web 09/03/2008
Metal-Triggered Radial Self-Assembly of Collagen Peptide Fibers
David E. Przybyla and Jean Chmielewski*
Department of Chemistry, Purdue UniVersity, 560 OVal DriVe, West Lafayette, Indiana 47907
Received June 27, 2008; E-mail: chml@purdue.edu
Collagen is one of the major components of the extracellular
matrix and encompasses skin, bone, tendons, ligaments, and blood
vessels. Collagen fibers are composed of bundled triple helices
consisting of three repeating Xaa-Yaa-Gly strands, where Pro-
HypGly (Hyp ) (2S,4R)-4-hydroxyproline) is the most frequently
observed unit. Currently collagen has multiple applications in cell
adhesion, tissue regeneration, and drug delivery.1 As a result, there
is an interest in creating synthetic collagen fibers that can be used
not only to mimic native collagen but also to enhance its biological
roles.2
One such approach has been to generate small collagen peptides
that self-assemble into collagen fibers. To date, the reported
strategies for generating these self-assembling collagen fibers have
employed linear growth through incorporation of a variety of N-
and C-terminal sticky ends. Specifically, electrostatic interactions,3
π-π stacking,4 a modified cysteine knot,5 and native chemical
ligation6 have been implemented. While these strategies have been
successful in generating collagen peptide fibers, there is still a need
to control the three-dimensional architecture of collagen networks,
a major necessity for tissue engineering. With this in mind, we
sought a means for incorporating an additional dimension of growth
into collagen peptide fibers.
In this report, we describe a design for metal-triggered radial
growth of a collagen triple-helix into collagen fibers (Figure 1).
The metal trigger was implemented because of its versatility in
other polypeptide systems and known ability to control the structure
in metal-organic structures.7 The design relies on a repeating
sequence of POG with a bipyridyl-modified lysine residue in place
of the hydroxyproline residue in the central tripeptide (Figure 1a,
H-byp). We hypothesized that this single amino acid modification
would still allow for stable collagen triple helix formation on the
basis of numerous host-guest collagen peptide studies.8 The
position of the three bipyridyl ligands in the center of the triple
helix yields three potential directions for radial growth (Figure 1b).
Upon the addition of metal ions, multiple triple helices can self-
assemble in a radial direction, potentially yielding three-dimensional
collagen networks (Figure 1c).
The peptide H-byp was synthesized by standard solid-phase
synthesis on a ChemMatrix rink amide resin via HBTU coupling
using Lys(Mtt)-OH in the central position. The removal of the Mtt
protecting group was performed on the solid support with DCM/
TFA (98:2), and the free amine was subsequently coupled with
4′-methyl-2,2′-bipyridine-4-carboxylic acid. The peptide was cleaved
from the resin with TFA/TIPS/H2O (95:2.5:2.5), purified to
homogeneity by reverse phase HPLC, and characterized by
MALDI-TOF mass spectroscopy.
The circular dichroism (CD) spectrum of H-byp was examined
to determine if the peptide formed a stable triple helix and to
investigate the effect of added metal ions. The CD spectrum of
H-byp (250 µM) displayed a typical collagen triple helix profile
with a maximum at 225 nm, and addition of metal ion, such as
Fe(II), had no effect on the CD spectrum, confirming that a triple
Figure 1. Collagen-mimetic peptide, triple helix, and metal-triggered
assembly: (a) amino acid sequence of H-byp; (b) side view of H-byp after
triple helix formation (peptide, red; bipyridine modification, blue); (c) top
view of a single triple helix followed by metal-triggered assembly.
helix also formed under these conditions (see Supporting Informa-
tion). Thermal denaturation studies were performed with H-byp
to determine the stability of its triple helix. Although somewhat
less stable then (POG)9 (Tm of 67 °C, data not shown), a Tm of 56
°C was observed for H-byp (see Supporting Information). However,
in the presence of the metal ion Fe(II), the Tm increased to 63 °C.
This increase in thermal stability with added metal ion could be
attributed to metal-promoted assembly of multiple triple helixes or
to intrastrand coordination within a single triple helix. Furthermore,
upon the addition of EDTA (100 mM) the Tm returned to 57 °C,
indicating that the enhanced metal-promoted stability was also
reversible.
UV-vis titration was used to confirm the presence of a complex
between H-byp and Fe(II), and determine the binding stoichiometry
(Figure 2a). It has previously been established that the metal to
ligand charge transfer resulting from the binding of bipyridine with
Fe(II) generates an absorbance maximum of 540 nm.9 The addition
of Fe(II) to a H-byp solution (54 µM) generated a magenta solution
with a maximum absorbance at 540 nm. A maximum absorbance
was observed at a relative molar ratio of 1:3 Fe(II):H-byp,
consistent with the bidentate coordination of bipyridine to octahedral
Fe(II) (Figure 1c).
Dynamic light scattering (DLS) was used to determine if the
addition of Fe(II) created larger ordered species with the collagen
peptide (Figure 2b). A solution of H-byp (1 mM) was preheated
to 70 °C followed by addition of Fe(II) (0.5 mM) and 4 day
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2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. TEM image of H-byp (2 mM), Fe(II) (0.3 mM), in HEPES (10
mM pH 7.0).
within the CTHs, and these sticky ends could promote fiber
formation. This fiber morphology was found to be lost at higher
concentrations of Fe(II) (1 mM) (see Supporting Information),
presumably because of the saturation of the bipyridyl ligand with
metal ions limiting peptide assembly and fiber growth.
In summary, we have introduced the concept of collagen fiber
formation through the radial growth of a collagen mimetic peptide,
H-byp, via a metal-assisted self-assembling trigger. We demonstrate
that the fiber formation was triggered through the addition of Fe(II)
and the assembly consisted of branched fibers at concentrations
below the ligand-metal binding stoichiometry. This system serves
as a proof of principle that collagen fibers can be initiated via
nonlinear assembly, and that modifications along the collagen
backbone may yield a new class of biologically active fibers. Future
studies will investigate how the ligand placement and coordination
chemistry can control the architectures of the collagen fibers.
Figure 2. (a) UV-vis titration of H-byp (54 µM) with Fe(II); (b) dynamic
light scattering of H-byp (1 mM), in 10 mM HEPES pH 7.0 (black), with
Fe(II) (500 µM) (red), and with Fe(II) (500 µM) and EDTA (100 mM)
(green).
incubation at 20 °C. A hydrodynamic radius of approximately 3
nm was observed for H-byp, consistent with other collagen triple
helical peptides.4 However, in the presence of Fe(II) a broad
distribution of radii were observed with a mean radius of 500 nm.
This suggested that the presence of Fe(II) facilitated the assembly
of larger aggregates. At lower peptide concentrations (250 and 50
µM) larger assemblies were also observed (mean radii of 200 and
150 nm, respectively) in conjuction with monomeric triple helices
(see Supporting Information). Next, we sought to investigate the
reversibility of the assembly by adding the metal chelator EDTA.
The data indicated that the assembly was completely reversible and
the assembled peptide returned to its monomeric triple helix. A
screen of other metal ions, such as Cu(II), Zn(II), and Ni(II) (see
Supporting Information) was also performed to determine if the
assembly process was specific to different metal ions. Of these,
Cu(II) was also found to promote the assembly of H-bpy.
Acknowledgment. We are grateful for financial support from
the NSF (Grant 0078923-CHE).
Supporting Information Available: Additional experimental details
and figures. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Transmission electron microscopy (TEM) was used to visualize
the morphology of the assembled H-byp-Fe(II) species. A solution
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addition of Fe(II) (0.3 mM) and a 4 day incubation at 4 °C (Figure
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