Importance of ring puckering versus interstrand hydrogen bonds for the conformational stability of collagen

Article abstract of DOI:10.1002/anie.201008118

Mismatch is fine: Proline derivatives with a ring pucker mismatching that of natural collagen but with favorable torsional angles along the peptide chain are readily tolerated within the collagen triple helix (see picture). In contrast, a competition between intramolecular and interstrand H bonds destabilizes the collagen triple helix. Copyright

Full text of DOI:10.1002/anie.201008118

DOI: 10.1002/anie.201008118
Collagen
Importance of Ring Puckering versus Interstrand Hydrogen Bonds for
the Conformational Stability of Collagen**
Roman S. Erdmann and Helma Wennemers*
The fibrous protein collagen is the most abundant protein in
mammals and plays a crucial role both in numerous cellular
[1]
activities and as a structural protein. Understanding the
factors that govern the conformational stability of collagen is
therefore important. In addition, there is a growing interest in
collagen-based functional materials and as a result the
development of synthetic collagen that bears functionalizable
[2,3]
groups is important.
Collagen is built up of single strands that form triple
[1]
helices which then further assemble into bundles and fibres.
The single strands consist of repeating Xaa–Yaa–Gly units
with all amide bonds in trans conformation. Proline (Pro) is
most often found in the Xaa position and (4R)-hydroxypro-
line (Hyp) in the Yaa position. Within the triple helix the
three strands are held together by hydrogen bonds between
the NH group of glycine (Gly) of one strand and the C=O
group of Pro of the adjacent strand (Figure 1a). Crystal
structures show C(4)-endo ring puckers of the Pro residues in
the Xaa and C(4)-exo ring puckers of the Hyp residues in the
[1,4]
Yaa positions (Figure 1a).
For the dihedral angles Y (Ni-
C -C -Ni+1) that are responsible for the directionality of the
ia
i
Figure 1. a) General structure of collagen. b) Ac–(4S)Acp–OCH with
C(4)-endo conformation by intramolecular H bonding.
3
collagen strands average values of approximately 1558 (Xaa)
[
1,4]
and approximately 1508 (Yaa) are observed.
Studies with collagen model peptides (CMPs) in which the
natural Pro and Hyp residues were replaced by other proline
derivatives led to the conclusion that both the ring puckering
and the interstrand H bonds are crucial for the conforma-
gen bonding leads to a preference for the C(4)-endo ring
pucker and the trans amide conformer in aqueous solutions
(Figure 1b). This is due to an enforced dihedral angle Y at
[
5,6]
[7]
tional stability of the collagen triple helix. All of the studies
that address the importance of the ring puckering were
performed with proline derivatives in which the trans amide
conformer is significantly favored over the cis conformer in
the case of C(4)-exo ring-puckered derivatives, whereas the
trans conformer is less favored in derivatives with C(4)-endo
approximately 1408 by the intramolecular H bond, which
allows for a stabilization of the trans isomer by a n!p*
[
7]
interaction. This dihedral angle Y within (4S)Acp is
comparable to those observed in collagen and proline
[1d]
derivatives such as (4R)Hyp with a C(4)-exo ring pucker.
[3,5]
ring puckers.
In addition, the Y angles of the C(4)-endo
Proline derivatives such as (4S)Acp therefore allow for the
first time to investigate, whether a C(4)-endo ring pucker is
tolerated in the Yaa position without a concomitant unfa-
vorable bias on the trans amide bond and the Y angle.
Furthermore, the combination of the C(4)-endo ring pucker
and the intramolecular H bond allows for probing whether
the ring pucker or the interstrand H bonds are more
important in the Xaa position for the stability of the collagen
triple helix. Herein, we demonstrate that a mismatched ring
pucker is tolerated, whereas the interstrand H bonds are
crucial for the conformational stability of the collagen triple
helix.
ring puckered derivatives examined so far are typically
approximately 1808 and are not close to those in collagen.
Thus, any C(4)-endo ring-puckered derivative had an unfa-
vorable bias towards the trans amide bond and the Y angle.
We have recently introduced proline derivatives, such as
(
4S)-acetamido proline (Acp), in which intramolecular hydro-
[
*] R. S. Erdmann, Prof. Dr. H. Wennemers
Department of Chemistry, University of Basel
St. Johanns-Ring 19, 4056 Basel (Switzerland)
Fax: (+41)61-267-0976
We started our investigations by analyzing the possible
effects of the incorporation of (4S)Acp in the Yaa position on
the properties of the collagen triple helix (Figure 2): The ring
pucker of (4S)Acp is C(4)-endo and a mismatch to that of the
C(4)-exo pucker of the natural (4R)Hyp residue. Thus, if the
E-mail: helma.wennemers@unibas.ch
[
**] This work was supported by the NCCR NANO and Bachem. H.W. is
grateful to Bachem for an endowed professorship.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201008118.
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6835

Communications
C(4)-exo ring puckering in the Yaa
does not destabilize the collagen triple helix to a significant
extent. It shows that a C(4)-exo ring pucker is not strictly
required in the Yaa position of collagen. An overlay of
(4S)Acp with a (4R)Hyp residue in the crystal structure of a
collagen triple helix shows the mismatched puckering. It also
illustrates that the Y angles of (4S)Acp and the natural
collagen strand are in good agreement and that no steric
constraints arise (Figure 4). In addition, the overlay shows
position is crucial for the stability of
the collagen triple helix, incorporation
of (4S)Acp in the Yaa position should
lead to a significant destabilization.
To probe the effect experimentally
we prepared CMP 1Y bearing (4S)Acp
in the Yaa position of the middle repeat
[
4b]
Figure 2. Structural
differences between
a (4S)Acp residue
[8]
unit within a 21mer. In addition, CMP
Y that bears the conformationally
(
black) and the natu-
ral (4R)Hyp residue
gray) in the Yaa
position of collagen.
2
analogous but sterically different (4S)-
(
formamido proline (Fmp) in the Yaa
[7,9]
position was prepared.
Such host–
guest CMPs with one varied residue
have proven valuable for the investiga-
tion of the effect of single residues on the conformational
[10]
stability of collagen. CMPs 3 and 4 with (4R)Hyp and Pro
residues, respectively, in the Yaa position were prepared for
comparison.
Figure 4. Overlay of the lowest-energy structure of Ac-(4S)Acp-OMe
[
7]
(
gray) with a (4R)Hyp residue (orange) in the Yaa position of
[
4b]
collagen (PDB 1 V7H).
that the intramolecular H bond within the (4S)Acp residue
does not interfere with the interstrand H bonds that hold the
[
12]
Thermal denaturation studies using circular dichroism
three strands together.
(
CD) spectroscopy as a monitoring tool were used to
To further analyze the importance of the ring puckering
for the stability of the collagen triple helix, we also
incorporated (4S)Acp and (4S)Fmp in the Xaa position and
investigate the relative stabilities of the collagen triple helices
derived from CMPs 1Y, 2Y, 3, and 4. All CMPs formed triple
helices as indicated by the observed maxima at 225 nm, which
[13]
prepared CMPs 1X and 2X.
[
1,5]
is typical for the collagen triple helix.
Upon heating
solutions of 1Y, 2Y, 3, and 4, comparable midpoints of
thermal transition (T ) were observed (Figure 3, spheres and
m
triangles).
For the known reference compounds 3 and 4, T values of
m
4
38C and 408C, respectively, were observed, which are
[
3,11]
consistent with previous studies.
The Tm value of CMPs
In the Xaa position, the C(4)-endo ring puckering of
(4S)Fmp and (4S)Acp matches that of the natural Pro residue
(Figure 5a). However, since the carbonyl group of Pro is
involved in the interstrand H bonds of collagen, the intra-
molecular H bond within (4S)Acp and (4S)Fmp can be
1
Y and 2Y are 408C and 398C, respectively, and identical or
close to those of CMPs 4 and 3. These results demonstrate
that the C(4)-endo ring puckering of (4S)Amp and (4S)Acp
Figure 5. a) Structural differences between a (4S)Acp residue (black)
and the natural Pro residue (gray) in the Xaa position of collagen.
b) Overlay of the lowest-energy structure of Ac-(4S)Acp-OMe (gray)
[
7]
Figure 3. CD thermal denaturation curves for CMPs 1Y (~), 2Y (~), 3
with a Pro residue (pink) in the Xaa position of collagen (PDB
1V7H).
[4b]
(
*), 4 (*), 1X (
&
), and 2X (&), in aqueous 50 mm AcOH.
6
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Angew. Chem. Int. Ed. 2011, 50, 6835 –6838

expected to compete with these interstrand H bonds that hold
the three collagen strands together. Incorporation of a
reflects at least in part the hindered formation of the three
interstrand H bonds because of the engagement of the
carbonyl groups in the intramolecular H bonds within the
(4S)Acp and (4S)Fmp residues. The thermodynamic param-
eters of the collagen triple helix derived from CMP 1Y and
2Y are comparable to those of the parent CMP 4. This result
demonstrates that the enthalpic and entropic contributions of
residues with C(4)-endo puckering do not differ significantly
from those of residues with C(4)-exo puckering. The slight
differences in the enthalpy and entropy for the triple helix
formation observed for CMPs 1Yand 2Yare likely caused by
either differences in the solvation properties of formamido
(
4S)Acp residue in the Xaa position will therefore allow for
comparing the importance of the ring pucker relative to that
of the interstrand H bonds.
CD-spectroscopic studies demonstrate that also CMPs 1X
and 2X form collagen triple helices. However, thermal
denaturation studies demonstrate that the stability of the
triple helices derived from 1X and 2X (Figure 3, squares) are
significantly lower (T ꢀ 308C) compared to those of CMPs
m
1
Y, 2Y, 3, and 4. Thus, despite the matching ring puckering of
(
4S)Acp and (4S)Fmp in the Xaa position the collagen triple
[
16]
helices derived from 1X and 2X are significantly destabilized.
An overlay of the (4S)Acp residue with a Pro residue in the
Xaa position of collagen illustrates that this destabilization
can be easily explained by the interference of the intra-
molecular H bond within (4S)Acp or (4S)Fmp with the
versus acetamido groups
or steric effects, which will be
further explored in future studies.
In conclusion, we have shown that a C(4)-endo ring
pucker is tolerated in the Yaa position of collagen triple
helices. This result shows that the ring puckering is less
important for the stability of collagen, provided that the
dihedral angles Y and the trans/cis amide conformer ratio
favor the formation of a triple helix. Furthermore, we have
demonstrated that the interstrand H bonds are significantly
more important for the stability of the collagen triple helix
than the ring puckering. The results are not only important for
the basic understanding of the factors that determine the
stability of collagen but also provide insight into which
positions of collagen can be derivatized with functional
groups without significantly disturbing the stability. Such
functionalized collagen model peptides are becoming increas-
[
14]
interstrand H bond (Figure 5b). The results show that the
formation of interstrand H bonds is significantly more
important for the stability of the collagen triple helix than
the ring puckering.
The observed Tm values provide insight into the relative
stabilities of the collagen triple helices. To gain a deeper
understanding of the conformational stabilities of the colla-
gen triple helices derived from CMPs 1Y, 1X, 2Y, and 2X, we
performed additional thermal denaturation experiments at a
lower heating rate and also monitored the refolding process.
From the resulting data, the thermodynamic parameters were
derived using a model introduced by Bꢀchinger, Engel, and
[
2]
ingly important as biocompatible functional materials.
[
15]
co-workers (see the Supporting Information).
The free energies (DG) of the triple helices derived from
the CMPs reflect the order observed in the melting temper-
atures (Table 1). Whereas the free energies of the triple
Received: December 22, 2010
Revised: May 3, 2011
Published online: June 8, 2011
Keywords: cis/trans amide bonds · collagen · PPII helix · proline ·
protein engineering
.
Table 1: Thermodynamic parameters for the thermal denaturation of the
CMPs.
[
a]
Entry
CMP
Tm
DH
[kcalmol
ꢁTDS
DG
[kcalmol ]
[
b]
ꢁ1
ꢁ1
ꢁ1 [c]
[
C]
]
[kcalmol
]
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Angew. Chem. Int. Ed. 2011, 50, 6835 –6838
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www.angewandte.org
6837

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3
6
838
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