Báo cáo khoa học: Effect of the -Gly-3(S)-hydroxyprolyl-4(R)-hydroxyprolyltripeptide unit on the stability of collagen model peptides ppt

Effect of the -Gly-3(S)-hydroxyprolyl-4( R)-hydroxyprolyl-
tripeptide unit on the stability of collagen model peptides
Kazunori Mizuno
1
, David H. Peyton
2
, Toshihiko Hayashi
3
,Ju
¨
rgen Engel
4
and Hans Peter
Ba
¨
chinger
1,5
1 Research Department, Shriners Hospital for Children, Portland, OR, USA
2 Department of Chemistry, Portland State University, Portland, OR, USA
3 Faculty of Pharmaceutical Science, Teikyo Heisei University, Chiba, Japan
4 Biozentrum, University of Basel, Switzerland
5 Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA
The collagen triple helix is probably the most abun-
dant protein motif in the human body. It comprises
three left-handed polyproline II-like helices with a
Gly-Xaa-Yaa repeat. These form a right-handed super
helix with a one-residue stagger [1,2]. The collagen
triple helix has many unique properties. One of them
is the requirement for many post-translational
modifications to produce the final tissue form of the
molecule [3]. In vertebrate collagens, most of the Pro

residues in the Yaa position of the -Gly-Xaa-Yaa-
repeat sequence are nearly completely 4-hydroxylated
to 4(R)-hydroxyproline [4(R)Hyp] by the enzyme prol-
yl 4-hydroxylase (EC 1.14.11.2). This modification in
the Yaa position is strongly related to the stability of
the collagen triple helix. Prolyl 4-hydroxylation also
occurs in the Xaa position in invertebrates. In addition
to prolyl 4(R)-hydroxylation, a small numbers of pro-
line residues are modified to 3(S)-hydroxyproline
[3(S)Hyp] [4,5] in many types of vertebrate collagens,
such as types I, II, III, IV, V and X. Invertebrate col-
lagens also contain 3(S )Hyp, for example interstitial
and cuticle collagens of annelids [6], crab sub-cuticular
Keywords
3-hydroxylation; collagen; peptide; post-
translational modification; thermal stability
Correspondence
H. P. Ba
¨
chinger, Research Department,
Shriners Hospital for Children, 3101 SW
Sam Jackson Park Road, Portland, OR
97239, USA
Fax: +1 503 221 3451
Tel: +1 503 221 3433
E-mail:
Website: />(Received 31 July 2008, revised 18
September 2008, accepted 25 September
2008)
doi:10.1111/j.1742-4658.2008.06704.x

In order to evaluate the role of 3(S)-hydroxyproline [3(S)-Hyp] in the
triple-helical structure, we produced a series of model peptides with nine
tripeptide units including 0–9 3(S)-hydroxyproline residues. The sequences
are H-(Gly-Pro-4(R)Hyp)
l
-(Gly-3(S)Hyp-4(R)Hyp)
m
-(Gly-Pro-4(R)Hyp)
n
-
OH, where (l, m, n) = (9, 0, 0), (4, 1, 4), (3, 2, 4), (3, 3, 3), (1, 7, 1) and (0,
9, 0). All peptides showed triple-helical CD spectra at room temperature
and thermal transition curves. Sedimentation equilibrium analysis showed
that peptide H-(Gly-3(S)Hyp-4(R)Hyp)
9
-OH is a trimer. Differential scan-
ning calorimetry showed that replacement of Pro residues with 3(S)Hyp
residues decreased the transition enthalpy, and the transition temperature
increases by 4.5 °C from 52.0 °C for the peptide with no 3(S)Hyp residues
to 56.5 °C for the peptide with nine 3(S)Hyp residues. The refolding kin-
etics of peptides H-(Gly-3(S)Hyp-4(R)Hyp)
9
-OH, H-(Gly-Pro-4(R)Hyp)
9
-
OH and H-(Gly-4(R)Hyp-4(R )Hyp)
9
-OH were compared, and the apparent
reaction orders of refolding at 10 °C were n = 1.5, 1.3 and 1.2, respec-
tively. Replacement of Pro with 3(S)Hyp or 4(R)Hyp has little effect on

the refolding kinetics. This result suggests that the refolding kinetics of
collagen model peptides are influenced mainly by the residue in the Yaa
position of the -Gly-Xaa-Yaa- repeated sequence. The experiments indicate
that replacement of a Pro residue by a 3(S)Hyp residue in the Xaa position
of the -Gly-Xaa-4(R)Hyp- repeat of collagen model peptides increases the
stability, mainly due to entropic factors.
Abbreviations
CRTAP, cartilage-associated protein; DSC, differential scanning calorimetry; Hyp, hydroxyproline; P3H1, prolyl 3-hydroxylase 1.
5830 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS
collagen [7], lobster sub-cuticular membrane collagen
[7], squid skin collagen [7], abalone muscle collagen [7],
octopus skin collagen [8], octopus arm collagen [9] and
jellyfish mesogloea collagen [10]. Most earlier reports
on 3(S)Hyp depended on amino acid analysis of pep-
tide fragments from crude extracts or whole tissues.
Only a few studies have determined the position of
3(S)Hyp by amino acid sequencing, and the 3(S)Hyp
was found in a -Gly-3Hyp-4Hyp-Gly- sequence in all
instances. Prolyl 3-hydroxylation is catalyzed by the
enzyme prolyl-3-hydroxylase (P3H1, EC 1.14.11.7),
which has three family members in vertebrates, but
characterization of these enzymes is very limited [11–
13]. The analysis of the 3(S)Hyp content is not
straightforward [14]. 3(S)Hyp degrades much faster
than 4(R)Hyp during hydrolysis in 6 m HCl, as used in
amino acid analysis [14]. In fact, reported contents of
3(S)Hyp are inconsistent even for the same tissue and
species [15–17]. This is not just due to the heterogene-
ity of the modification, but also to differences in sam-
ple preparation for amino acid analysis. As a result of

this, some reports may have underestimated the
3(S)Hyp content.
Type I collagen, which consists of two a1 chains
and one a2 chain, has a single 3(S)Hyp residue per
chain [18–20]. The proline residue at position 986 in
the a1 chain is modified to 3(S)Hyp [21] by the protein
complex P3H1 ⁄ CRTAP ⁄ cyclophilin B [13]. Post-trans-
lational modifications are changed in heritable disor-
ders due to mutation and ⁄ or deletion of the enzymes,
or due to over-modification, as in osteogenesis imper-
fecta and Ehlers–Danlos syndrome type VI [22].
The 4(R)-hydroxylation in the Yaa position has been
well documented as stabilizing the triple-helical struc-
ture [23–25]. Raines and colleagues [23] synthesized
fluoroprolyl compounds containing C-F bonds, a very
weak hydrogen bond acceptor [26], in order to deter-
mine the mechanism of stabilization. The increase in
stability is due to a stereoelectronic effect (reviewed in
[23]). The effect of 4(R)Hyp in the Xaa position of the
-Gly-Xaa-Yaa- collagen sequence has also been ana-
lyzed [23,27,28]. Compared to the post-translational
modification at the C4 position, the effect of 3-hydrox-
ylation of prolyl residues in the collagen helix on its
stability has not yet been thoroughly analyzed [29–31].
Whether the 3(S)Hyp residue in the Xaa position
stabilizes or destabilizes the collagen helix is still
controversial. In host-guest peptides, it was found that
the stability of the triple helix is decreased when Pro in
the Xaa position is replaced by either 3(S)Hyp or
3(S)fluoroproline [30,31]. It is not possible for 3(S)Hyp

to be located in the Yaa position in the triple-helical
structure due to steric clashes [31].
Recently, we analyzed the crystal structure of a triple-
helical peptide with two 3(S)Hyp residues per chain, i.e.
H-(Gly-Pro-4(R)Hyp)
3
-(Gly-3(S)Hyp-c4(R)Hyp)
2
-(Gly-
Pro-4(R)Hyp)
4
-OH [29]. The backbone of this peptide is
almost identical to that of triple-helical peptides com-
prising the repeated sequences Gly-Pro-Pro and Gly-
Pro-4(R)Hyp in a left-handed 7 ⁄ 2 helical symmetry [32].
This finding led us to re-evaluate the data obtained
using the peptides acetyl-(Gly-3(S)Hyp-4(R)Hyp)
10
-
NH
2
and acetyl-(Gly-Pro-4(R)Hyp)
3
-Gly-3(S)Hyp-
4(R)Hyp-(Gly-Pro-4(R)Hyp)
4
-Gly-Gly-NH
2
[33]. The
peptide acetyl-(Gly-3(S)Hyp-4(R)Hyp)

10
-NH
2
did not
show any evidence of forming a triple helix when ana-
lyzed by sedimentation-equilibrium, CD or NMR ana-
lysis [33]. We repeated the synthesis of this peptide and
also produced several other peptides with 3(S)Hyp in
the Xaa position. All of these newly synthesized peptides
formed a triple-helical structure. We confirmed that a
peptide with 3(S)Hyp in the Yaa position, acetyl-(Gly-
Pro-3(S)Hyp)
10
-NH
2
, does not fold into a triple-helical
structure. We analyzed the peptides by CD, NMR and
differential scanning calorimetry (DSC) to evaluate
the effect of 3(S)-hydroxylation on the stability and
refolding kinetics of the collagen triple helix.
Results
The CD spectra of the newly synthesized peptide
Ac-(Gly-3(S)Hyp-4(R)Hyp)
10
-NH
2
in water shown in
Fig. 1 is similar to that of other collagen-like peptides.
The spectrum shows a positive peak around 225 nm
×

4°C
4°C
80 °C
80 °C
Fig. 1. CD spectra of acetyl-(Gly-3(S)Hyp-4(R)Hyp)
10
-NH
2
.CD
spectra were measured at 4, 20, 40 and 80 °C in water at a con-
centration of 100 l
M. The positive ellipticity at 225 nm decreases
as the temperature is increased from 4 to 20, 40 and 80 ° C.
K. Mizuno et al. 3-hydroxyproline in the collagen triple helix
FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5831
and a negative peak around 196 nm at 4 °C. The
ellipticity at 225 nm of peptide Ac-(Gly-3(S)Hyp-
4(R)Hyp)
10
-NH
2
is between that of peptide Ac-(Gly-
Pro-Pro)
10
-NH
2
and Ac-(Gly-Pro-4(R)Hyp)
10
-NH
2

,
and larger than the other collagen-like peptides which
do not form a triple helix, Ac-(Gly-4(R)Hyp-Pro)
10
-
NH
2
and Ac-(Gly-Pro-3(S)Hyp)
10
-NH
2
[33]. The
temperature scan monitored at 225 nm shows a
cooperative transition curve for peptide Ac-(Gly-
3(S)Hyp-4(R)Hyp)
10
-NH
2
(Fig. 2). The ellipticity of
the peptide was positive even after the transition
at 95 °C. Therefore, the characteristics of peptide
Ac-(Gly-3(S)Hyp-4(R)Hyp)
10
-NH
2
are similar to those
of peptide Ac-(Gly-4(R)Hyp-4(R)Hyp)
10
-NH
2

[34].
In order to verify that this transition curve is due to
the transition from triple helix to coil, the oligomeriza-
tion state of the 3(S)Hyp-containing peptides was ana-
lyzed by equilibrium sedimentation (Fig. S1). Analysis
of peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)
10
-NH
2
showed
a molecular mass of 8.75 ± 0.15 · 10
3
Da; the
calculated molecular mass of the trimer peptide is
8676 Da. We also analyzed H-(Gly-3(S)Hyp-4(R)
Hyp)
9
-OH. The molecular mass for this peptide is
8.26 ± 0.21 · 10
3
Da, which is 7% larger than the
calculated trimeric peptide value of 7703 Da, suggest-
ing that most of the peptide is trimeric and that some
aggregates are in solution at 25 °C. We conclude from
these experiments that the collagen model peptide with
repeated tripeptide units -Gly-3(S)Hyp-4(R)Hyp- forms
a trimer in aqueous solution. The triple-helical nature
of Gly-3(S)Hyp-4(R)Hyp is also supported by a previ-
ously determined crystal structure [29].
In our previous paper [33], we used the 3(S)-Hyp

commercially available from Fluka (Buchs, Switzer-
land). Amino acid analysis and MALDI-TOF mass
spectroscopy showed the expected molecular weight
(2892 Da). However, this peptide did not form a triple
helix. We attempted to determine why the previously
used peptide did not form a triple helix. The source of
the 3(S)Hyp from Fluka that we used previously was
hydrolyzed bovine collagen. No information about the
preparation of the commercial product is available
from the company, and the product is not available in
the USA. 3(S)-hydroxyproline is known to degrade
faster and isomerize to 3(R)Hyp more easily than
4(R)Hyp to 4(S)Hyp under acidic conditions [14].
Therefore, hydrolysis of collagen could lead to the
isomerization of 3(S)Hyp. We used the method of
Bellon et al. [14] involving labeling with 4-chloro-7-
nitro-2,1,3-benzoxadiazole labeling and also labeling
with 4-fluoro-7-nitrobenzofurazan to detect potential
isomers of 3(S)Hyp, such as 3(R)Hyp and the d-iso-
mer. Unfortunately, the same batch of product that we
used previously was no longer available and we did
not have enough original peptide left for this analysis.
We could not detect a significant amount of 3(R)Hyp
by thin-layer chromatography using a different batch
of 3(S)Hyp from Fluka. The peptide acetyl-(Gly-
3(S)Hyp-4(R)Hyp)
10
-NH
2
has thirty 3(S)Hyp residues

in the triple-helical structure. If we assume that the
presence of one incorrect 3(S)Hyp in the middle six
tripeptide units causes the inability to form a triple
helix, a 10% incorrect isomer content in the 3(S)Hyp
preparation would mean that only 15% of the peptide
could form a triple helix. We assume that the 3(S)Hyp
batch from Fluka that we used for the first preparation
of the peptide contained a significant amount of isom-
erized 3(S)Hyp, but we do not have enough peptide
left to verify this hypothesis. However, the results
obtained by others [31] are consistent with this
assumption.
The transition temperature (T
m
) of peptide Ac-(Gly-
3(S)Hyp-4(R)Hyp)
10
-NH
2
was determined by CD in
H
2
O at 235 nm at a concentration of 2 mm peptide
with a heating rate of 7.5 °CÆh
)1
(Fig. 3A). The T
m
of
the peptide is 79.7 °C. Under the same conditions, the
T

m
is a little higher than that of peptide Ac-(Gly-Pro-
4(R)Hyp)
10
-NH
2
(76.1 °C) and very close to that of
Temperature (°C)
×
Fig. 2. Thermal transition curves of collagen-like peptides. The pep-
tides were measured in water and the CD signal was monitored at
225 nm as a function of increasing temperature. The peptide con-
centration was 100 l
M and the temperature scanning rate was
10 °CÆh
)1
. Results are shown for Ac-(Gly-3(S)Hyp-4(R) Hyp)
10
-NH
2
(square), Ac-(Gly-Pro-Pro)
10
-NH
2
(circle), Ac-(Gly-Pro-4(R)-Hyp)
10
-NH
2
(upwards triangle), Ac-(Gly-4(R)Hyp-Pro)
10

-NH
2
(downwards triangle)
and Ac-(Gly-Pro-3(S)Hyp)
10
-NH
2
(diamond). All data except those for
Ac-(Gly-3(S)Hyp-4(R) Hyp)
10
-NH
2
and Ac-(Gly-Pro-3(S)Hyp)
10
-NH
2
are
from a previous study [34] and are included as a reference.
3-hydroxyproline in the collagen triple helix K. Mizuno et al.
5832 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS
peptide Ac-(Gly-4(R)Hyp-4(R)Hyp)
10
-NH
2
(80.5 °C)
[34]. The transition curve of peptide Ac-(Gly-3(S)Hyp-
4(R)Hyp)
10
-NH
2

was not as sharp as that for Ac-(Gly-
Pro-4(R)Hyp)
10
-NH
2
. In order to analyze the thermo-
dynamic properties, the peptides were analyzed by
DSC in water (Fig. 3B). Peptide Ac-(Gly-3(S)Hyp-
4(R)Hyp)
10
-NH
2
has a smaller transition enthalpy than
peptide Ac-(Gly-Pro-4(R)Hyp)
10
-NH
2
, but a slightly
larger transition enthalpy than Ac-(Gly-4(R)Hyp-
4(R)Hyp)
10
-NH
2
[34]. The transition enthalpies and
entropies are summarized in Table 1.
Figure 4 shows the proton NMR spectra of
Ac-(Gly-4(R)Hyp-4(R)Hyp)
10
-NH
2

, Ac-(Gly-Pro-4(R)
Hyp)
10
-NH
2
and Ac-(Gly-3(S)Hyp-4( R)Hyp)
10
-NH
2
.
In each of these three spectra, the large line-widths
and the strong negative NOE cross-peaks are indica-
tive of strong triple-helix formation. Also, the reso-
nances at approximately 3.1–3.4 p.p.m. are markers
for triple-helix formation as noted previously [34,35].
This is further illustrated by its loss at high tempera-
tures, shown on the left of Fig. 4. The fact that the
line-widths are even greater in the Ac-(Gly-3(S)Hyp-
4(R)Hyp)
10
-NH
2
spectrum may indicate a different
degree of internal motions available to this species
compared with the others. Nevertheless, all of the
spectra are characteristic of collagen triple helices.
Refolding of peptide H-(Pro-4(R)Hyp-Gly)
10
-OH is
two orders of magnitude faster than that of peptide

H-(Pro-Pro-Gly)
10
-OH [36]. In order to assess the
effect of 3(S)Hyp in the refolding kinetics, the peptides
H-(Gly-3(S)Hyp-4(R)Hyp)
9
-OH, H-(Gly-Pro-4(R)Hyp)
9
-
OH and H-(Gly-4(R)Hyp-4(R)Hyp)
9
-OH were ana-
lyzed. Refolding was monitored by CD at 225 nm in a
concentration range from 2.7 · 10
)2
mm to 1.0 mm at
10 °C. The simple apparent initial reaction order [36]
was calculated as shown below:
d½H
dt

t¼0
¼ kC½
n
0
where [C]
0
is the initial peptide concentration, [H]is
the concentration of triple-helical molecules, k is the
rate constant, t is time, and n is the reaction order.

Temperature (°C)
(KJ·K
–1
·mol
–1
)
Temperature (°C)
Fig. 3. (A) Thermal transition curves of collagen-like peptides. The
peptides were measured in water, and the CD signal was moni-
tored at 235 nm as a function of temperature. The peptide concen-
tration was 2 m
M and the temperature scanning rate was
7.5 °CÆh
)1
. Results are shown for Ac-(Gly-3(S)Hyp-4(R) Hyp)
10
-NH
2
(square), Ac-(Gly-Pro-4(R)-Hyp)
10
-NH
2
(upwards triangle) and
Ac-(Gly-4(R)-Hyp-4(R)-Hyp)
10
-NH
2
(circle). Both heating and cooling
scans are shown. The open symbols indicate heating scans, and
the filled symbols indicate cooling scans. (B) Differential scanning

calorimetry of collagen-like peptides. The peptides were dissolved
in water, and scanned at 7.5 °CÆh
)1
. Results are shown for Ac-(Gly-
3(S)Hyp-4(R) Hyp)
10
-NH
2
(solid line), Ac-(Gly-Pro-4(R)-Hyp)
10
-NH
2
(dashed line) and Ac-(Gly-4(R)-Hyp-4(R)-Hyp)
10
-NH
2
(dotted line).
Both heating scans (positive values) and cooling scans (negative
values) are shown. The rate of temperature change is 7.5 °CÆh
)1
in
both directions. The data for peptides Ac-(Gly-Pro-4(R) Hyp)
10
-NH
2
and Ac-(Gly-4(R)Hyp-4(R)-Hyp)
10
-NH
2
are from a previous study [34]

and are included as a reference.
Table 1. Thermodynamic values for the thermal transitions of
Ac-(Gly-Xaa-Yaa)
l0
-NH
2
peptides. °, standard state for enthalpy and
entropy change.
DH°
(kJÆmol
)1
trimer)
DS
o
(JÆ°C
)1
Æ
mol
)1
trimer)
T
m
(°C)
Ac-(Gly-3(S)Hyp-4(R)Hyp)
10
-NH
2
Heating )207 )482 80.2
Cooling )220 )520 79.3
Ac-(Gly-Pro-4(R)Hyp)

10
-NH
2
Heating )337 )858 75.7
Cooling )323 )823 74.3
Ac-(Gly-4(R)Hyp-4(R)Hyp)
10
-NH
2
Heating )169 )385 81.8
Cooling )168 )371 79.8
K. Mizuno et al. 3-hydroxyproline in the collagen triple helix
FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5833
As the fraction folded, F, is defined as the fraction
of the peptide that forms a triple helix, F =3[H] ⁄ [C]
0
,
the equation
dF
dt

t¼0
¼ 3kC½
nÀ1
0
can be written as the logarithmic function
log
dF
dt


t¼0
¼ðn À 1Þ log C½
0
þconst
The apparent reaction order, n, can be obtained from
the slope, (n ) 1), when the logarithm of the initial
rate (dF ⁄ dt)
t=0
is plotted on the y axis, and the loga-
rithm of the total peptide concentration [C]
0
is plotted
on the x axis (Fig. 5). The apparent reaction orders of
peptides H-(Gly-Pro-4(R)Hyp)
9
-OH, H-(Gly-3(S)Hyp-
4(R)Hyp)
9
-OH and H-(Gly-4(R)Hyp-4(R)Hyp)
9
-OH
were n = 1.3, 1.5 and 1.2, respectively. These values
are similar to the value n = 1.5 obtained for peptide
H-(Pro-4(R)Hyp-Gly)
10
-OH at 7 °C [36]. Given the
solubility of the peptides and the detection limits of
the CD signals for analysis, it is virtually impossible to
acquire data for higher or lower concentrations.
Within the measured concentration range, these three

peptides refold much faster than peptide H-(Pro-Pro-
Gly)
10
-OH [36], implying that the 4(R)-hydroxyproline
in the Yaa position contributes most to the folding
rate of the model peptides, regardless of the Xaa posi-
tion imino acid modification, i.e. Pro, 3( S)Hyp or
4(R)Hyp. The large difference in the rates of the
H-(Pro-Pro-Gly)10-OH peptide and the other peptides
at low concentrations indicates the importance of
4(R)Hyp in the nucleation process. Future studies will
be performed to study the mechanism of refolding of
these model peptides in detail.
In order to evaluate the thermodynamic properties of
3(S)Hyp-containing collagen model peptides, a series of
peptides with nine tripeptide units comprising -Gly-Pro-
4(R)Hyp- and -Gly-3(S)Hyp-4(R)Hyp- were synthesized,
with the following sequences: H-(Gly-Pro-4(R )Hyp)
9
-
OH, H-(Gly-Pro-4(R)Hyp)
4
-Gly-3(S)Hyp-4(R)Hyp-(Gly-
Pro-4(R)Hyp)
4
-OH, H-(Gly-Pro-4(R)Hyp)
3
-(Gly-3(S)
Hyp-4(R)Hyp)
2

-(Gly-Pro-4(R)Hyp)
4
-OH, H-(Gly-Pro-
4(R)Hyp)
3
-(Gly-3(S)Hyp-4 (R)Hyp)
3
-(Gly-Pro-4(R)
Hyp)
3
-OH, H-(Gly-Pro-4(R)Hyp)
1
-(Gly-3(S)Hyp-4(R)
Hyp)
7
-(Gly-Pro-4(R)Hyp)
1
-OH and H-(Gly-3(S)Hyp-
4(R)Hyp)
9
-OH. The peptides were dissolved in NaCl ⁄ P
i
Fig. 4.
1
H-NMR spectra of collagen-like pep-
tides. Right: 2D NOESY data set for (A)
Ac-(Gly-3(S)Hyp-4(R)Hyp)
10
-NH
2

, (B) Ac-(Gly-
Pro-4(R)Hyp)
10
-NH
2
and (C) Ac-(Gly-4(R)Hyp-
4(R)Hyp)
10
-NH
2
, respectively. Left: Variable
temperature
1
H-NMR spectra for Ac-(Gly-
3(S)Hyp-4(R)Hyp)
10
-NH
2
at 75, 85, 95, 100
and 105 °C (from the bottom). Loss of the
3.2 p.p.m. peak at high temperatures is con-
sistent with the concomitant loss of triple-
helix content. Unless otherwise stated, the
spectra were recorded at 30 °C, 10 m
M
concentration, and the NOESY mixing time
was 60 ms. The 2D NOESY data sets were
processed using 60° phase-shifted sine bells
before Fourier transformation, and the 1D
spectra were treated with a 1 Hz line-broad-

ening factor.
3-hydroxyproline in the collagen triple helix K. Mizuno et al.
5834 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS
and analyzed by CD at 235 nm with a scanning rate of
6 °CÆh
)1
. All peptides showed a cooperative transi-
tion, and little hysteresis was observed, as the heating
and cooling curves almost overlapped under these
experimental conditions. As the number of 3(S)Hyp
in the peptide increases, the slope of the transition
becomes more shallow (Fig. 6), indicative of a
decrease in the transition enthalpy. The set of pep-
tides was also analyzed by DSC with a scanning rate
of 0.1–2.0 °CÆmin
)1
. More than four repeating cycles
of heating and cooling scans yielded overlapping
curves, indicating that folding and unfolding is a
reversible reaction under these conditions. The transi-
tion temperatures of the heating and cooling scans
were different with different scanning rates, but the
scanning rate had no effect on the transition enthalpy
for any peptide. Figure 7 shows the excess heat
capacity of the peptides as a function of temperature.
Replacement of Pro by 3(S)Hyp decreases the transi-
tion enthalpy. The first replacement of Pro by
3(S)Hyp in the middle of the triple helix has a large
effect on the transition enthalpy. Adding another also
shows a further significant decrease of the transition

enthalpy. Further additions only lead to a minor
decrease in the transition enthalpies. The numerical
data are given in Table 2.
Discussion
Our new experimental data indicate that a Pro to
3(S)Hyp modification in the Xaa position of -Gly-
Xaa-Yaa- collagen-like peptides increases the stability
of the triple-helical structure by a small margin.
Insertion of 3(S)Hyp in the context of the nine
tripeptide units increases the T
m
of the peptides by
approximately 0.5 °C per single replacement of -Gly-
Pro-4(R)Hyp- by -Gly-3(S)Hyp-4(R)Hyp Previously,
we reported the crystal structure of the triple helix of
peptide H(Gly-Pro-4(R)Hyp)
3
-(Gly-3(S)Hyp-4(R)Hyp)
2
-
(Gly-Pro-4(R)Hyp)
4
-OH [29]. This structure is almost
identical to the structure of other triple-helical (Gly-
Pro-Pro)
n
or (Gly-Pro-4(R)Hyp)
n
peptides. The height
per tripeptide unit and the 7 ⁄ 2 symmetry were similar

to those of other collagen peptides with imino acids in
both the Xaa and Yaa positions [32]. However, our
previous analysis of the stability of Ac-(Gly-3(S)Hyp-
4(R)Hyp)
10
-NH
2
seems inconsistent with this structure.
We therefore determined whether the presence of
acetyl and amide groups in this peptide prevented
triple-helical folding.
Jenkins et al. [31] analyzed the host-guest peptide
(Pro-4(R)Hyp-Gly)
3
-3(S)Hyp-4(R)Hyp-Gly-(Pro-4(R )
Hyp-Gly)
3
-OH, and reported that the Pro to 3(S)Hyp
Fig. 5. Refolding kinetics of peptides analyzed by CD. Refolding of
the collagen model peptides was monitored by CD at 225 nm. The
logarithm of the initial rate of triple-helix formation, log (dF ⁄ dt)
t =0
,
is plotted as a function of the logarithm of the total polypeptide
chain concentration, log [C]
0
. The peptides H-(Gly-3(S)Hyp-
4(R)Hyp)
9
-OH (filled circle), H-(Gly-Pro-4(R)Hyp)

9
-OH (filled upwards
triangle) and H-(Gly-4(R)Hyp-4(R)Hyp)
9
-OH (filled square) were mea-
sured at 10 °C. The values for peptides H-(Pro-4(R)Hyp-Gly)
10
-OH
(open upwards triangle) and H-(Pro-Pro-Gly)
10
-OH (open downwards
triangle) measured at 7 °C are from a previous study [36] and are
included for comparison.
10 20 30 40 50 60 70 80
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
3
2
1
9
[θ]

235 nm
(deg cm
2
·dmol
–1
× 10
–3
)
Temperature (°C)
0
7
3
1
Fig. 6. Thermal transition curves of a series of 3(S)Hyp-containing
peptides. Peptides H-(Gly-Pro-4(R)Hyp)
l
-(Gly-3(S)Hyp-4(R)Hyp)
m
-(Gly-
Pro-4(R)Hyp)
n
-OH, where (l, m, n) = (9, 0, 0), (4, 1, 4), (3, 2, 4), (3,
3, 3), (1, 7, 1) or (0, 9, 0), were analyzed by CD in NaCl ⁄ P
i
at a
peptide concentration of 2 m
M. The CD signal was monitored at
235 nm with a heating rate of 6 °CÆh
)1
.

K. Mizuno et al. 3-hydroxyproline in the collagen triple helix
FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5835
modification lowered the T
m
of the peptide by 3.3 °C.
When 3(S)fluoroproline was incorporated, a further
decrease in the T
m
was found [30]. As the pK
a
of the
carboxyl group of 3(S)Hyp is lower than that of Pro,
these authors suggested that the hydrogen bond
between the amide group of Gly and the carbonyl
group of 3(S)Hyp might be weaker than the hydrogen
bond between the amide group of Gly and the carbonyl
group of Pro. They also analyzed the crystal structure
of 3(S)Hyp-derived N-(
13
C
2
-acetyl)-3(S)-hydroxy-l-pro-
line methyl ester, and the structure of the pyrrolidine
ring is different from that of the N-(
13
C
2
-acetyl)-4(R)-
hydroxy-l-proline methyl ester. We are not sure
whether the lower T

m
found in the host-guest peptide
in their study is due to contamination or differences in
the methods of observing the CD transition. Our DSC
analysis showed that peptide Ac-(Gly-3(S)Hyp-
4(R)Hyp)
10
-NH
2
has a smaller transition enthalpy than
peptide Ac-(Gly-Pro-4(R)Hyp)
10
-NH
2
(Fig. 3B). The
smaller DH observed may be explained in several ways.
One is that the carbonyl group of 3(S)Hyp in the Xaa
position is a weak hydrogen bond acceptor, as
suggested by previous experimental data [31], because
the carboxyl pK
a
value for 3(S)Hyp (1.62) is lower than
that for Pro (1.92). The hydrogen bond between the
amide of Gly and the carbonyl group of the residues in
the Xaa position is the only direct inter-chain hydrogen
bond in the triple helix. Another explanation is that
there is probably a difference in hydration of the
unfolded chains. The peptide with 3(S)Hyp could
be more hydrated than the peptide with Pro in single
chains, which would cause a decrease in the transition

enthalpy and a decrease in the entropy of solvent water
Temperature (°C)
T
m
(°C)
ΔS (J·K
–1
·mole
–1
trimer)
Table 2. Thermodynamic values for the thermal transitions of
H-(Gly-Pro-4(R)Hyp)
l
-(Gly-3(S)Hyp-4(R)Hyp)
m
-(Gly-Pro-4(R)Hyp)
n
-OH
peptides.
Number of 3(S)
Hyp per peptide
DH°
(kJÆmol
)1
trimer)
DS°
(JÆ°C
)1
Æmol
)1

trimer) T
m
(°C)
a
0 )321 )881 52.0
1 )224 )581 52.8
2 )177 )439 51.0
3 )170 )418 52.6
7 )158 )373 56.2
9 )156 )370 55.3
a
Data from heating scans at a scanning rate of 0.5 °CÆmin
)1
.
Fig. 7. Differential scanning calorimetry of a series of 3(S)Hyp-con-
taining peptides. (A) Peptides H-(Gly-Pro-4(R)Hyp)
l
-(Gly-3(S)Hyp-
4(R)Hyp)
m
-(Gly-Pro-4(R)Hyp)
n
-OH, where (l, m, n) = (9, 0, 0), (4, 1,
4), (3, 2, 4), (3, 3, 3), (1, 7, 1) or (0, 9, 0), were analyzed by DSC at
2m
M peptide concentration in NaCl ⁄ P
i
. The excess heat capacity is
shown as a function of temperature with a scanning rate of
0.5 °CÆmin

)1
. (B) Transition enthalpy per mole trimer (left axis) and
the transition entropy (right axis) as a function of the number of
3(S)Hyp residues per chain. (C) Transition temperatures (T
m
)asa
function of the number of 3(S)Hyp residues per chain, fitted using
linear regression.
3-hydroxyproline in the collagen triple helix K. Mizuno et al.
5836 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS
molecules. Kawahara et al. [37] hypothesized that the
difference in the degree of hydration explains the
stability of the triple-helical structure of peptide
H-(4(R)Hyp-4(R)Hyp-Gly)
10
-OH. Recently, we analyzed
the density of peptides with the repeated sequence (Gly-
4(R)Hyp-4(R)Hyp)
n
and (Gly-Pro-4(R)Hyp)
n
(n = 5
and 9) over a wide range of temperature, and also
analyzed the solution structure of these peptides by
small-angle X-ray scattering (SAXS) [38]. Our data
indicate that at high temperatures, i.e. unfolded chains,
the peptides (Gly-4(R)Hyp-4(R)Hyp)
9
and (Gly-Pro-
4(R)Hyp)

9
have no significant structural differences.
Based on partial specific volume measurements, it is
suggested that the hydration number for the peptide
(Gly-Pro-4(R)Hyp)
9
increases with formation of the
triple-helix whereas that for the peptide (Gly-4(R)Hyp-
4(R)Hyp)
9
decreases. It is possible that peptides with
3(S)Hyp in the Xaa position also have these properties,
and therefore a smaller transition enthalpy.
The change in the transition enthalpy in the series of
host-guest 27-residue peptides was greatest for the first
replacement of Pro by 3(S)Hyp. The effect of
additional substitutions of Pro by 3(S)Hyp was smaller
than for the first substitution (Fig. 7). Two junctions
between Gly-Pro-4(R)Hyp and Gly-3(S)Hyp-4(R)Hyp
are introduced by the first addition of a 3(S)Hyp
residue, and this number remains constant upon
addition of further 3(S)Hyp residues. Therefore, the
first introduction of a 3(S)Hyp residue probably
changes the cooperativity and thermodynamic values
more strongly than further additions.
We can rule out the possibility that absence of the
3(S)Hyp residue in type I collagen affects the stability
of the triple helix, and stability or lack thereof is not
the reason for the phenotypes observed when
3(S)Hyp is missing. Mutations in P3H1 cause a form

of lethal osteogenesis imperfecta [39], and knockout
of the CRTAP gene encoding cartilage-associated
protein in mouse causes a severe osteogenesis imper-
fecta-like phenotype [40]. The proline at position 986
was not hydroxylated in the CRTAP null mice, as
analyzed by mass spectroscopy. However, it is
not clear how the absence of 3(S)Hyp residues in
type I collagen can cause these phenotypes. We have
ruled out stability as a factor. It is much more likely
that 3(S)Hyp takes part in protein–collagen
interactions required for bone formation. 3-hydroxyl-
ation of the proline at position 986 in the a1 chain
of type I collagen involves the protein complex
P3H1 ⁄ CRTAP ⁄ cyclophilin B [13,40]. All functions of
this complex in the rough endoplasmic reticulum need
to be considered when analyzing the observed pheno-
types. Is the phenotype only due to absence of the
single 3(S)Hyp in the a1 chains of type I collagen or
are other functions impaired as a result of mutations
in the molecules of the complex? It may well be that
3(S)Hyp is important for bone mineralization, but
other factors cannot be ruled out. Bone protein and
mineral interactions with type I collagen need to be
further characterized to identify protein–collagen
interactions that are affected by the lack of 3(S)Hyp
in type I collagen.
Experimental procedures
Peptide synthesis
Peptides were synthesized using an ABI 433A synthe-
sizer (Applied Biosystems, Foster City, CA, USA).

Couplings were performed using Fmoc-PAL-PEG-PS resin
(0.16 mmolÆg
)1
) (Applied Biosystems) for the C-terminal
amide-capped peptides, or O -t-butyl-l-trans-4-hydroxypro-
line-2-chlorotrityl resin (0.48 meqÆ g
)1
) (AnaSpec, San Jose,
CA, USA) for peptides with 4(R)Hyp at the C-terminal
end. Fmoc-amino acids Fmoc-Gly-OH and Fmoc-Pro-OH
were purchased from Applied Biosystems, Fmoc-4(R)
Hyp(tBu)-OH was purchased from Novabiochem (EMD
Biosciences Inc., San Diego, CA, USA), and acetyl
glycine was purchased from Bachem (Torrance, CA,
USA). Commercially available Fmoc-3(S)-hydroxyproline
(AnaSpec) was used without any further purification.
HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (Applied Biosystems) (4.0 eq)
and di-isopropylethylamine were used as the coupling
reagents for the Fmoc solid-phase peptide synthesis. The
peptides were cleaved from the resin using Reagent R
(trifluoroacetic acid⁄ thioanisole ⁄ 1,2-ethanedithiol ⁄ anisole,
90 : 5 : 3 : 2) at room temperature for 3 h. Peptides were
isolated by precipitation from the cleavage cocktail with
diethyl ether at 4 °C, dissolved in 0.1% trifluoroacetic
acid and purified by preparative HPLC using a Vydac
Ò
218TP101550 C18 column (5 lm internal diameter, 300 A
˚
pore size, 50 · 250 mm) and a 218TP15202503 guard

column (W.R. Grace & Co., Columbia, MD, USA) with
a flow rate of 36 mLÆmin
)1
and elution with a 0–50%
acetonitrile gradient in 0.1% trifluoroacetic acid. All
peptides were characterized by electrospray ⁄ quadrupole ⁄
time-of flight mass spectrometry (Q-tof micro, Waters
Corp., Milford, MA, USA), and amino acid analysis. The
Ac-(Gly-Pro-3(S)Hyp)
10
-NH
2
peptide was also characterized
by MALDI-TOF at the Stanford Protein and Nucleic Acid
facility (Stanford, CA, USA). The Ac-(Gly-3(S)Hyp-
4(R)Hyp)
10
-NH
2
peptide was analyzed by MALDI-TOF at
the Department of Dentistry, Oregon Health and Science
University (Portland, OR). The peptides were stored at
)20 °C before preparing stock solutions. The stock solutions
for analysis were stored at 4 °C.
K. Mizuno et al. 3-hydroxyproline in the collagen triple helix
FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5837
Circular dichroism
CD spectra were recorded on an Aviv 202 spectropolari-
meter (Aviv Biomedical Inc., Lakewood, NJ, USA) using a
Peltier thermostatted cell holder and a 1 mm path-length

rectangular quartz cell (Starna Cells Inc., Atascadero, CA,
USA). Peptide concentrations were determined by amino
acid analysis (L-8800A amino acid analyzer; Hitachi High
Technologies America Inc., San Jose, CA, USA). The
wavelength spectra represent the mean of at least 10 scans,
with 0.1 nm resolution of one second averaged data. The
temperature scanning experiments were run at 0.1 °CÆmin
)1
.
For determination of refolding kinetics, the peptides were
denatured at 75 °C for 10 min, and then rapidly diluted at
ratios from 1 : 4 to 1 : 64 with solvent cooled on ice. After
rapid mixing, the sample solution was immediately put into
the 1 mm path-length rectangular quartz cuvette cell in the
CD spectrometer holder pre-incubated at 10 °C. The ellip-
ticity at 225 nm was monitored as a function of time. The
fraction of folded peptide (F) is defined as
F ¼
h
obs
À h
DN
h
N
À h
DN
where h
obs
, h
DN

and h
N
represent the observed, monomeric
and triple-helical peptide ellipticity, respectively. h
N
was
measured directly at the temperature used for refolding
(10 °C). h
DN
was determined by linear extrapolation of the
straight line measured under denatured conditions between
75 and 85 °C.
Analytical ultracentrifugation
Sedimentation-equilibrium analysis was performed using a
Beckman Coulter ProteomeLabÔ model XL-A analytical
ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA,
USA). The An-60 Ti rotor was used together with 12 mm
Epon centerpiece double-sector cells with quartz windows.
The peptides were analyzed in 20 mm phosphate buffer,
pH 7.2, containing 150 mm NaCl, unless otherwise indi-
cated. The peptide concentrations were adjusted from 0.02
to 0.1 mgÆmL
)1
. Sedimentation equilibrium measurements
were performed at temperatures of 4 or 25 °C. The analysis
was performed using scientist software (Micromath,
St Louis, MO, USA) with the assumption that there is a
single molecular species in the solution. A partial specific
volume of 0.67 mLÆg
)1

was used for all calculations.
Differential scanning calorimetry
Differential scanning calorimetry was performed using a
Nano II model 6100 differential scanning calorimeter
(Calorimetry Science Corporation) with 0.299 mL capil-
lary cells. A stock sample solution in water was prepared
and adjusted to the required peptide concentration in the
sample solution. The peptide sample in NaCl ⁄ P
i
was
de-gassed before analysis. The heating and cooling scans
of the peptides used in this experiment were all reprodu-
cible in several repeat scans. The heating and cooling
rates ranged between 0.1 and 2.0 °CÆmin
)1
. The data were
analyzed using cpcalc software (Calorimetry Science
Corporation, Lindon, UT, USA). The DC
p
values for
the peptides analyzed in this paper were assumed to be
zero. All the heat-capacity curves were fitted using
the polynomial baseline fit. The concentration of the
peptide was determined by amino acid analysis.
NMR spectroscopy
NMR spectra were recorded on a Bruker AMX-400 spec-
trometer operating at 400.14 MHz (Bruker, Madison, WI,
USA). The 90° pulse width was 9 ls, and a low-power 2 s
presaturation pulse was applied to suppress the H
2

O
(HOD) resonance. The spectra were recorded as 16 384
points for the 1D spectra, and as 1024 · 512 data point sets
for the 2D spectra. NOESY data were collected with time
proportional phase increment in the indirect dimension, at
mixing times between 30 and 120 ms, and with a total
recording time of approximately 10 h. TOCSY data were
collected with various mixing times, ranging from 30
to 90 ms. The data were processed using swan-mr to
1024 · 1024 real data sets after application of a 60° phase-
shifted sin
2
function and Fourier transformation for the 2D
spectra; baselines were straightened using polynomials as
required. Spectra were referenced to 0 p.p.m. via internal
2,2-dimethylsilapentane-5-sulfonate or via the water
resonance (4.71 p.p.m. at 30 °C). Final visualization and
analyses of the 2D data sets were performed using
nmrview [41] or swan-mr [42].
Acknowledgements
This work was supported by a grant from Shriners
Hospital for Children (Portland, OR, USA). The
authors thank Eric A. Steel and Jessica L. Hacker for
expert technical assistance and Dr B. Kerry Maddox
for amino acid analyses.
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Supporting information
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Fig. S1. Sedimentation equilibrium analysis of colla-
gen-like peptides containing 3(S)Hyp.
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3-hydroxyproline in the collagen triple helix K. Mizuno et al.
5840 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS