Metal-assisted assembly and stabilization of collagen-like triple helices

Article abstract of DOI:10.1021/ja0442062

Single-chain and TRIS-assembled collagen mimetic peptide structures incorporating catechol groups were synthesized. When 1/3 equiv of Fe³⁺ was added to the single-chain compound in 50 mM CAPS buffer (pH 10), the 1:3 Fe³⁺-catechol complex that formed acted as an N-terminal scaffold to assemble the triple helix. When 1 equiv of Fe³⁺ was added to the TRIS-assembled compound in the buffer solution, the Fe³⁺-catechol complex acted as an extra C-terminal scaffold, which lead to a triple helix with both termini tethered. The formation of this C-terminal complex increased the T_m by a remarkable 22 °C! Copyright

Full text of DOI:10.1021/ja0442062

Published on Web 11/02/2004
Metal-assisted Assembly and Stabilization of Collagen-like Triple Helices
Weibo Cai, Sen Wai Kwok, Joseph P. Taulane,* and Murray Goodman†
Department of Chemistry and Biochemistry, UniVersity of California-San Diego, La Jolla, California 92093
Received September 23, 2004; E-mail: jpt@chem.ucsd.edu
Collagen is the most abundant extracellular protein in vertebrates.
It has a unique triple helical motif in which three polypeptide chains
form left-handed helices and are supercoiled into a right-handed
triple helix.1,2 The primary sequence of triple helical collagen is
composed of Gly-Xaa-Yaa trimer repeats.3,4 A variety of scaffolds
have been utilized to facilitate the assembly of collagen-like triple
helices.5-10 In particular, Koide and co-workers reported the
assembly and stabilization of a collagen-like triple helix by an
N-terminal Fe(II)-bipyridine complex.10 Here, we report the
incorporation of an Fe3+-catechol complex as a scaffold to
assemble peptide chains into triple helices.
Enterobactin, a naturally occurring triscatechol, is the most
powerful Fe3+ chelator ever reported with an overall stability
constant of 1049.11,12 Therefore, we have chosen to use the Fe3+
-
catechol complex as a scaffold because of the robust nature of the
complex. The Fe3+-catechol complex has been well studied. A
1:3 Fe3+-catechol complex is formed at a pH above 9.5, which
has a characteristic absorption peak at around 500 nm (for the
octahedrally coordinated Fe3+) in the UV-vis spectra.13,14
Two molecules incorporating the catechol moiety have been
synthesized (Figure 1). In the single-chain compound (A), a
dopamine residue was attached to the N-terminus of the peptide
chain composed of the Gly-Nleu-Pro sequence (where Nleu denotes
N-isobutylglycine) via a flexible succinic acid linker. In the TRIS-
assembled peptide (B), 2,3-dihydroxybenzoic acid was attached to
the C-terminus via an ethylenediamine linker. The structure of the
TRIS scaffold is noted in Figure 1.15 The linkers were incorporated
to accommodate the one amino acid residue register between the
three strands of a triple helix.16 Molecules A and B have the same
peptide sequence and chain length.
The UV-vis spectra of the molecules with and without Fe3+ in
50 mM (pH 10) CAPS buffer are shown in Figures 2 and 3 (where
CAPS denotes 3-cyclohexylamino-1-propane sulfonic acid). Fresh,
but nondegassed, solutions were stored under N2, and no oxidation
of the catechol was observed. In the case of compound A, 1/3 equiv
of Fe3+ was added. For compound B, 1 equiv of Fe3+ was added
since one molecule of B contains three catechol groups. Both spectra
exhibit the broad absorbance band at around 500 nm, which clearly
suggests octahedral coordination of Fe3+ (red wine color).
The triple helicity of all of the structures were determined by
thermal denaturation monitored by optical rotation and circular
dichroism (CD) measurements (Figures 4 and 5). All of the
measurements for compound A were carried out at 0.2 mg/mL,
while all of the measurements for compound B were carried out at
0.1 mg/mL due to a solubility problem. Since catechol, in the
absence of Fe3+, is easily oxidized at elevated temperatures, the
thermal denaturation measurements for both compounds without
Fe3+ were carried out in 1 mM HCl to prevent oxidation. Melting
curves for model compound Boc-â-Ala-TRIS-[(Gly-Nleu-Pro)6-
OMe]3 were obtained in 1 mM HCl (pH 3), in H2O (pH 7) and 50
mM CAPS buffer (pH 10), and they are superimposable with
Figure 1. Single-chain (A) and TRIS-assembled (B) peptides incorporating
catechol groups.
Figure 2. The UV-vis spectrum of compound A (0.2 mg/mL) with and
without Fe3+ in 50 mM CAPS buffer.
Figure 3. The UV-vis spectrum of compound B (0.1 mg/mL) with and
without Fe3+ in 50 mM CAPS buffer.
the same melting temperature (Tm) (see Supporting Information).
Therefore, the difference in triple helical stability between the
peptide without Fe3+ in 1 mM HCl and the peptide with Fe3+ in
50 mM CAPS buffer would only come from the Fe3+-catechol
complex.
For peptides composed of the Gly-Nleu-Pro sequence, the
positive peak in the CD spectrum is only present when it is triple
helical.17,18 The CD data shown in Figure 4 indicate that compound
A does not form a triple helix when Fe3+ is absent. This was further
† Professor Murray Goodman died on June 1, 2004.
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10.1021/ja0442062 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Figure 6. Schematic representation of the triple helix formed by compound
B when Fe3+ is added.
by an intramolecular complex where Fe3+ is coordinated by three
catechol groups from the same molecule B.
On the basis of the above data, we believe that the Fe3+-catechol
complex formed in each case acts as an extra scaffold to facilitate
the folding of the peptide into triple helices. For compound A, the
complex acts as an N-terminal scaffold. For compound B, the
complex acts as a C-terminal scaffold, which confers extra stability
to the TRIS-assembled triple helix. A schematic of this discaffold-
assembled triple helical collagen mimetic structure is shown in
Figure 6.
Figure 4. Circular dichroism spectra of A (dark green), A + Fe3+
(magenta), B (blue), and B + Fe3+ (red). All measurements were carried
out at 6 °C in 50 mM CAPS buffer (pH 10).
In conclusion, we report here the assembly of a triple helix which
is tethered at both the N- and the C-terminus by a TRIS scaffold
and an Fe3+-catechol complex, respectively. The Fe3+-catechol
complex raised the Tm of the TRIS-assembled triple helix by a
remarkable 22 °C. Further characterization of the discaffold-
assembled structures is currently underway.
Acknowledgment. This project is funded by the NSF Bioma-
terial Division, DMR0111617.
Supporting Information Available: Experimental details and
figures. This material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 5. Thermal melting transitions of A (dark green), A + Fe3+
(magenta), B (blue), and B + Fe3+ (red). Measurements without Fe3+ were
carried out in 1 mM HCl to prevent oxidation of the catechol groups.
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confirmed by the absence of a melting transition in the thermal
melting measurement (Figure 5). However, when 1/3 equiv of Fe3+
was added, the positive peak appears in the CD spectra. Thermal
melting measurement revealed that the Tm of the triple helix is 28
°C. Since the UV-vis spectrum indicates the presence of a 1:3
Fe3+-catechol complex, it is clear that this complex acts as a
scaffold to assemble the collagen-like triple helix. The triple helix
folding rate of compound A when Fe3+ is present is comparable to
the rate of scaffold-assembled peptides of the same sequence (data
not shown).
For TRIS-assembled peptide B, both CD and thermal melting
experiments indicate that the molecule is triple helical in solution
even when Fe3+ is not present. The melting temperature is 36 °C.
When 1 equiv of Fe3+ is added, the molecule exhibits an
extraordinary Tm of 58 °C. The formation of an Fe3+-catechol
complex increased the Tm of B by a remarkable 22 °C!
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â-Ala linker).19,20 An increase of 3-4 °C in the Tm value over the
corresponding TRIS-assembled structure would be observed.
However, an increase of 22 °C in the Tm value can only be explained
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