Differential scan-ning calorimetry showed that replacement of Pro residues with 3SHyp residues decreased the transition enthalpy, and the transition temperature increases by 4.5C from 52
Trang 1tripeptide unit on the stability of collagen model peptides Kazunori Mizuno1, David H Peyton2, Toshihiko Hayashi3, Ju¨rgen Engel4and Hans Peter
Ba¨chinger1,5
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-hydroxprol-ylase (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)-hydroxypropro-line [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: hpb@shcc.org
Website: http://www.shcc.org/bach_lab.htm
(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 4.5C from 52.0 C for the peptide with no 3(S)Hyp residues
to 56.5C 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 10C 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.
Trang 2collagen [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 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
-NH2 and acetyl-(Gly-Pro-4(R)Hyp)3 -Gly-3(S)Hyp-4(R)Hyp-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 [33] The peptide acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 did not show any evidence of forming a triple helix when 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-NH2, 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-NH2 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.
Trang 3and a negative peak around 196 nm at 4C The
ellipticity at 225 nm of peptide
Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 is between that of peptide
Ac-(Gly-Pro-Pro)10-NH2 and Ac-(Gly-Pro-4(R)Hyp)10-NH2,
and larger than the other collagen-like peptides which
do not form a triple helix, Ac-(Gly-4(R)Hyp-Pro)10
-NH2 and Ac-(Gly-Pro-3(S)Hyp)10-NH2 [33] The
temperature scan monitored at 225 nm shows a
cooperative transition curve for peptide
Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 (Fig 2) The ellipticity of
the peptide was positive even after the transition
at 95C Therefore, the characteristics of peptide
Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 are similar to those
of peptide Ac-(Gly-4(R)Hyp-4(R)Hyp)10-NH2[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-NH2 showed
a molecular mass of 8.75 ± 0.15· 103Da; 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· 103Da, 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-NH2 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 (Tm) of peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 was determined by CD in
H2O at 235 nm at a concentration of 2 mm peptide with a heating rate of 7.5 CÆh)1 (Fig 3A) The Tm of the peptide is 79.7 C Under the same conditions, the
Tmis a little higher than that of peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2 (76.1C) 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-NH2
(square), Ac-(Gly-Pro-Pro)10-NH2(circle), Ac-(Gly-Pro-4(R)-Hyp)10-NH2
(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-NH2and Ac-(Gly-Pro-3(S)Hyp)10-NH2are
from a previous study [34] and are included as a reference.
Trang 4peptide Ac-(Gly-4(R)Hyp-4(R)Hyp)10-NH2 (80.5C)
[34] The transition curve of peptide
Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2was not as sharp as that for
Ac-(Gly-Pro-4(R)Hyp)10-NH2 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-NH2has a smaller transition enthalpy than peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2, but a slightly larger transition enthalpy than Ac-(Gly-4(R)Hyp-4(R)Hyp)10-NH2 [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-NH2, Ac-(Gly-Pro-4(R) Hyp)10-NH2 and Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2
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-NH2 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)2mm to 1.0 mm at
10C The simple apparent initial reaction order [36] was calculated as shown below:
d½H
dt
t¼0
¼ k C½ n0
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)
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-NH2 (upwards triangle) and
Ac-(Gly-4(R)-Hyp-4(R)-Hyp)10-NH2(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-NH2 (dotted line).
Both heating scans (positive values) and cooling scans (negative
values) are shown The rate of temperature change is 7.5 CÆh)1in
both directions The data for peptides Ac-(Gly-Pro-4(R) Hyp)10-NH2
and Ac-(Gly-4(R)Hyp-4(R)-Hyp)10-NH2are 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
(JÆC)1Æ mol)1 trimer)
T m
(C) Ac-(Gly-3(S)Hyp-4(R)Hyp) 10 -NH 2 Heating )207 )482 80.2
Ac-(Gly-Pro-4(R)Hyp)10-NH2 Heating )337 )858 75.7
Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 Heating )169 )385 81.8
Trang 5As 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 3k C½ n10
can be written as the logarithmic function
log dF
dt
t¼0
¼ ðn 1Þ log C½ 0þconst
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 7C [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 modificaposi-tion, 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 Gly-Pro-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⁄ Pi
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-NH2, (B) Ac-(Gly-Pro-4(R)Hyp)10-NH2and (C) Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 , respectively Left: Variable temperature1H-NMR spectra for Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2at 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.
Trang 6and analyzed by CD at 235 nm with a scanning rate of
6CÆh)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 0.1–2.0CÆmin)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 Tm of the peptides by approximately 0.5C 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-NH2 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
] 235 nm
2 ·dmol
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.
Trang 7modification lowered the Tmof the peptide by 3.3C.
When 3(S)fluoroproline was incorporated, a further
decrease in the Tm was found [30] As the pKa 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-(13C2 -acetyl)-3(S)-hydroxy-l-pro-line methyl ester, and the structure of the pyrrolidine ring is different from that of the N-(13C2 -acetyl)-4(R)-hydroxy-l-proline methyl ester We are not sure whether the lower Tm 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-NH2has a smaller transition enthalpy than peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2 (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 pKavalue 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)
Tm
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
(JÆC)1Æmol)1
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
2 m 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 ) as a function of the number of 3(S)Hyp residues per chain, fitted using linear regression.
Trang 8molecules 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
Couplings were performed using Fmoc-PAL-PEG-PS resin
amide-capped peptides, or
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
(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
90 : 5 : 3 : 2) at room temperature for 3 h Peptides were isolated by precipitation from the cleavage cocktail with
218TP101550 C18 column (5 lm internal diameter, 300 A˚
column (W.R Grace & Co., Columbia, MD, USA) with
acetonitrile gradient in 0.1% trifluoroacetic acid All
Corp., Milford, MA, USA), and amino acid analysis The
by MALDI-TOF at the Stanford Protein and Nucleic Acid
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
Trang 9Circular 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
For determination of refolding kinetics, the peptides were
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
ellip-ticity at 225 nm was monitored as a function of time The
fraction of folded peptide (F) is defined as
measured directly at the temperature used for refolding
straight line measured under denatured conditions between
Analytical ultracentrifugation
Sedimentation-equilibrium analysis was performed using a
Beckman Coulter ProteomeLab 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
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
Differential scanning calorimetry
Differential scanning calorimetry was performed using a
(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
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
the peptides analyzed in this paper were assumed to be
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
(HOD) resonance The spectra were recorded as 16 384
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
spectra; baselines were straightened using polynomials as required Spectra were referenced to 0 p.p.m via internal
analyses of the 2D data sets were performed using
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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